Expression vectors for stimulating an immune response and methods of using the same

ABSTRACT

The present invention relates to nucleic acid vaccines encoding multiple CTL and HTL epitopes and MHC targeting sequences.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation and claims the benefit of Ser. No.09/078,904, filed May 13, 1998, now abandoned, and 60/085,751, filed May15, 1998;, now abandoned, both herein incorporated by reference in theirentirety.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

This invention was made with government support under NIH Grant No.AI-42699-01, NIH Grant No. AI38584-03, and NIH Contract No.N01-AI-45241. The Government has certain rights in this invention.

FIELD OF THE INVENTION SUBMISSION ON COMPACT DISC

The contents of the following submission on compact discs areincorporated herein by reference in its entirety: A compact disc copy ofthe substitute Sequence Listing (COPY 1) (file name: 3996320022.txt,date recorded: Jan. 4, 2002, size:199 KB); a duplicate compact disc copyof the substitute Sequence Listing (COPY 2) (file name: 3996320022.txt,date recorded: Jan. 4, 2002, size: 199 KB); a computer readable formcopy of the substitute Sequence Listing (CRF COPY) (file name:3996320022.txt, date recorded: Jan. 4, 2002, size: 199 KB).

The present invention relates to nucleic acid vaccines encoding multipleCTL and HTL epitopes and MHC targeting sequences.

BACKGROUND OF THE INVENTION

Vaccines are of fundamental importance in modern medicine and have beenhighly effective in combating certain human diseases. However, despitethe successful implementation of vaccination programs that have greatlylimited or virtually eliminated several debilitating human diseases,there are a number of diseases that affect millions worldwide for whicheffective vaccines have not been developed.

Major advances in the field of immunology have led to a greaterunderstanding of the mechanisms involved in the immune response and haveprovided insights into developing new vaccine strategies (Kuby,Immunology, 443-457 (3rd ed., 1997), which is incorporated herein byreference). These new vaccine strategies have taken advantage ofknowledge gained regarding the mechanisms by which foreign material,termed antigen, is recognized by the immune system and eliminated fromthe organism. An effective vaccine is one that elicits an immuneresponse to an antigen of interest.

Specialized cells of the immune system are responsible for theprotective activity required to combat diseases. An immune responseinvolves two major groups of cells, lymphocytes, or white blood cells,and antigen-presenting cells. The purpose of these immune response cellsis to recognize foreign material, such as an infectious organism or acancer cell, and remove that foreign material from the organism.

Two major types of lymphocytes mediate different aspects of the immuneresponse. B cells display on their cell surface specialized proteins,called antibodies, that bind specifically to foreign material, calledantigens. Effector B cells produce soluble forms of the antibody, whichcirculate throughout the body and function to eliminate antigen from theorganism. This branch of the immune system is known as the humoralbranch. Memory B cells function to recognize the antigen in futureencounters by continuing to express the membrane-bound form of theantibody.

A second major type of lymphocyte is the T cell. T cells also have ontheir cell surface specialized proteins that recognize antigen but, incontrast to B cells, require that the antigen be bound to a specializedmembrane protein complex, the major histocompatibility complex (MHC), onthe surface of an antigen-presenting cell. Two major classes of T cells,termed helper T lymphocytes (“HTL”) and cytotoxic T lymphocytes (“CTL”),are often distinguished based on the presence of either CD4 or CD8protein, respectively, on the cell surface. This branch of the immunesystem is known as the cell-mediated branch.

The second major class of immune response cells are cells that functionin antigen presentation by processing antigen for binding to MHCmolecules expressed in the antigen presenting cells. The processedantigen bound to MHC molecules is transferred to the surface of thecell, where the antigen-MHC complex is available to bind to T cells.

MHC molecules can be divided into MHC class I and class II molecules andare recognized by the two classes of T cells. Nearly all cells expressMHC class I molecules, which function to present antigen to cytotoxic Tlymphocytes. Cytotoxic T lymphocytes typically recognize antigen boundto MHC class I. A subset of cells called antigen-presenting cellsexpress MHC class II molecules. Helper T lymphocytes typically recognizeantigen bound to MHC class II molecules. Antigen-presenting cellsinclude dendritic cells, macrophages, B cells, fibroblasts, glial cells,pancreatic beta cells, thymic epithelial cells, thyroid epithelial cellsand vascular endothelial cells. These antigen-presenting cells generallyexpress both MHC class I and class II molecules. Also, B cells functionas both antibody-producing and antigen-presenting cells.

Once a helper T lymphocyte recognizes an antigen-MHC class II complex onthe surface of an antigen-presenting cell, the helper T lymphocytebecomes activated and produces growth factors that activate a variety ofcells involved in the immune response, including B cells and cytotoxic Tlymphocytes. For example, under the influence of growth factorsexpressed by activated helper T lymphocytes, a cytotoxic T lymphocytethat recognizes an antigen-MHC class I complex becomes activated. CTLsmonitor and eliminate cells that display antigen specifically recognizedby the CTL, such as infected cells or tumor cells. Thus, activation ofhelper T lymphocytes stimulates the activation of both the humoral andcell-mediated branches of the immune system.

An important aspect of the immune response, in particular as it relatesto vaccine efficacy, is the manner in which antigen is processed so thatit can be recognized by the specialized cells of the immune system.Distinct antigen processing and presentation pathways are utilized. Theone is a cytosolic pathway, which results in the antigen being bound toMHC class I molecules. An alternative pathway is an endoplasmicreticulum pathway, which bypasses the cytosol. Another is an endocyticpathway, which results in the antigen being bound to MHC class IImolecules. Thus, the cell surface presentation of a particular antigenby a MHC class II or class I molecule to a helper T lymphocyte or acytotoxic T lymphocyte, respectively, is dependent on the processingpathway for that antigen.

The cytosolic pathway processes endogenous antigens that are expressedinside the cell. The antigen is degraded by a specialized proteasecomplex in the cytosol of the cell, and the resulting antigen peptidesare transported into the endoplasmic reticulum, an organelle thatprocesses cell surface molecules. In the endoplasmic reticulum, theantigen peptides bind to MHC class I molecules, which are thentransported to the cell surface for presentation to cytotoxic Tlymphocytes of the immune system.

Antigens that exist outside the cell are processed by the endocyticpathway. Such antigens are taken into the cell by endocytosis, whichbrings the antigens into specialized vesicles called endosomes andsubsequently to specialized vesicles called lysosomes, where the antigenis degraded by proteases into antigen peptides that bind to MHC class IImolecules. The antigen peptide-MHC class II molecule complex is thentransported to the cell surface for presentation to helper T lymphocytesof the immune system.

A variety of factors must be considered in the development of aneffective vaccine. For example, the extent of activation of either thehumoral or cell-mediated branch of the immune system can determine theeffectiveness of a vaccine against a particular disease. Furthermore,the development of immunologic memory by inducing memory-cell formationcan be important for an effective vaccine against a particular disease(Kuby, supra). For example, protection from infectious diseases causedby pathogens with short incubation periods, such as influenza virus,requires high levels of neutralizing antibody generated by the humoralbranch because disease symptoms are already underway before memory cellsare activated. Alternatively, protection from infectious diseases causedby pathogens with long incubation periods, such as polio virus, does notrequire neutralizing antibodies at the time of infection but insteadrequires memory B cells that can generate neutralizing antibodies tocombat the pathogen before it is able to infect target tissues.Therefore, the effectiveness of a vaccine at preventing or amelioratingthe symptoms of a particular disease depends on the type of immuneresponse generated by the vaccine.

Many traditional vaccines have relied on intact pathogens such asattenuated or inactivated viruses or bacteria to elicit an immuneresponse. However, these traditional vaccines have advantages anddisadvantages, including reversion of an attenuated pathogen to avirulent form. The problem of reversion of an attenuated vaccine hasbeen addressed by the use of molecules of the pathogen rather than thewhole pathogen. For example, immunization approaches have begun toincorporate recombinant vector vaccines and synthetic peptide vaccines(Kuby, supra). Recently, DNA vaccines have also been used (Donnelly etal., Annu. Rev. Immunol. 15:617-648 (1997), which is incorporated hereinby reference). The use of molecules of a pathogen provides safe vaccinesthat circumvent the potential for reversion to a virulent form of thevaccine.

The targeting of antigens to MHC class II molecules to activate helper Tlymphocytes has been described using lysosomal targeting sequences,which direct antigens to lysosomes, where the antigen is digested bylysosomal proteases into antigen peptides that bind to MHC class IImolecules (U.S. Pat. No. 5,633,234; Thomson et al., J. Virol.72:2246-2252 (1998)). It would be advantageous to develop vaccines thatdeliver multiple antigens while exploiting the safety provided byadministering individual epitopes of a pathogen rather than a wholeorganism. In particular, it would be advantageous to develop vaccinesthat effectively target antigens to MHC class II molecules foractivation of helper T lymphocytes.

Several studies also point to the crucial role of cytotoxic T cells inboth production and eradication of infectious diseases and cancer by theimmune system (Byrne et al., J. Immunol. 51:682 (1984); McMichael etal., N. Engl. J. Med. 309:13 (1983)). Recombinant protein vaccines donot reliably induce CTL responses, and the use of otherwise immunogenicvaccines consisting of attenuated pathogens in humans is hampered, inthe case of several important diseases, by overriding safety concerns.In the case of diseases such as HIV, HBV, HCV, and malaria, it appearsdesirable not only to induce a vigorous CTL response, but also to focusthe response against highly conserved epitopes in order to preventescape by mutation and overcome variable vaccine efficacy againstdifferent isolates of the target pathogen.

Induction of a broad response directed simultaneously against multipleepitopes also appears to be crucial for development of efficaciousvaccines. HIV infection is perhaps the best example where an infectedhost may benefit from a multispecific response. Rapid progression of HIVinfection has been reported in cases where a narrowly focused CTLresponse is induced whereas nonprogressors tend to show a broaderspecificity of CTLs (Goulder et al., Nat. Med. 3:212 (1997); Borrow etal., Nat. Med. 3:205 (1997)). The highly variable nature of HIV CTLepitopes resulting from a highly mutating genome and selection by CTLresponses directed against only a single or few epitopes also supportsthe need for broad epitope CTL responses (McMichael et al., Annu. Rev.Immunol. 15:271 (1997)).

One potential approach to induce multispecific responses againstconserved epitopes is immunization with a minigene plasmid encoding theepitopes in a string-of-beads fashion. Induction of CTL, HTL, and B cellresponses in mice by minigene plasmids have been described by severallaboratories using constructs encoding as many as 11 epitopes (An etal., J. Virol. 71:2292 (1997); Thomson et al., J. Immunol. 157:822(1996); Whitton et al., J. Virol. 67:348 (1993); Hanke et al., Vaccine16:426 (1998); Vitiello et al., Eur. J. Immunol. 27:671-678 (1997)).Minigenes have been delivered in vivo by infection with recombinantadenovirus or vaccinia, or by injection of purified DNA via theintramuscular or intradermal route (Thomson et al., J. Immunol: 160:1717(1998); Toes et al., Proc. Natl. Acad. Sci. USA 94:14660 (1997)).

Successful development of minigene DNA vaccines for human use willrequire addressing certain fundamental questions dealing with epitopeMHC affinity, optimization of constructs for maximum in vivoimmunogenicity, and development of assays for testing in vivo potency ofmulti-epitope minigene constructs. Regarding MHC binding affinity ofepitopes, it is not currently known whether both high and low affinityepitopes can be included within a single minigene construct, and whatranges of peptide affinity are permissible for CTL induction in vivo.This is especially important because dominant epitopes can vary in theiraffinity and because it might be important to be able to delivermixtures of dominant and subdominant epitopes that are characterized byhigh and low MHC binding affinities.

With respect to minigene construct optimization for maximumimmunogenicity in vivo, conflicting data exists regarding whether theexact position of the epitopes in a given construct or the presence offlanking regions, helper T cell epitopes, and signal sequences might becrucial for CTL induction (Del Val et al., Cell 66:1145 (1991); Bergmannet al., J. Virol. 68:5306 (1994); Thomson et al., Proc. Natl. Acad. Sci.USA 92:5845 (1995); Shirai et al., J. Infect. Dis. 173:24 (1996);Rahemtulla et al., Nature 353:180 (1991); Jennings et al., Cell.Immunol. 133:234 (1991); Anderson et al., J. Exp. Med. 174:489 (1991);Uger et al., J. Immunol. 158:685 (1997)). Finally, regarding developmentof assays that allow testing of human vaccine candidates, it should benoted that, to date, all in vivo immunogenicity data of multi-epitopeminigene plasmids have been performed with murine class I MHC-restrictedepitopes. It would be advantageous to be able to test the in vivoimmunogenicity of minigenes containing human CTL epitopes in aconvenient animal model system.

Thus, there exists a need to develop methods to effectively deliver avariety of HTL (helper T lymphocyte) and CTL (cytotoxic T lymphocyte)antigens to stimulate an immune response. The present inventionsatisfies this need and provides related advantages as well.

SUMMARY OF THE INVENTION

The invention therefore provides expression vectors encoding two or moreHTL epitopes fused to a MHC class II targeting sequence, as well asexpression vectors encoding a CTL epitope and a universal HTL epitopefused to an MHC class I targeting sequence. The HTL epitope can be auniversal HTL epitope (also referred to as a universal MHC class IIepitope). The invention also provides expression vectors encoding two ormore HTL epitopes fused to a MHC class II targeting sequence andencoding one or more CTL epitopes. The invention additionally providesmethods of stimulating an immune response by administering an expressionvector of the invention in vivo, as well as methods of assaying thehuman immunogenicity of a human T cell peptide epitope in vivo in anon-human mammal.

In one aspect, the present invention provides an expression vectorcomprising a promoter operably linked to a first nucleotide sequenceencoding a major histocompatibility (MHC) targeting sequence fused to asecond nucleotide sequence encoding two or more heterologous peptideepitopes, wherein the heterologous peptide epitopes comprise two HTLpeptide epitopes or a CTL peptide epitope and a universal HTL peptideepitope.

In another aspect, the present invention provides a method of inducingan immune response in vivo comprising administering to a mammaliansubject an expression vector comprising a promoter operably linked to afirst nucleotide sequence encoding a major histocompatibility (MHC)targeting sequence fused to a second nucleotide sequence encoding two ormore heterologous peptide epitopes, wherein the heterologous peptideepitopes comprise two HTL peptide epitopes or a CTL peptide epitope anda universal HTL peptide epitope.

In another aspect, the present invention provides a method of inducingan immune response in vivo comprising administering to a mammaliansubject an expression vector comprising a promoter operably linked to afirst nucleotide sequence encoding a major histocompatibility (MHC)targeting sequence fused to a second nucleotide sequence encoding aheterologous human HTL peptide epitope.

In another aspect, the present invention provides a method of assayingthe human immunogenicity of a human T cell peptide epitope in vivo in anon-human mammal, comprising the step of administering to the non-humanmammal an expression vector comprising a promoter operably linked to afirst nucleotide sequence encoding a heterologous human CTL or HTLpeptide epitope.

In one embodiment, the heterologous peptide epitopes comprise two ormore heterologous HTL peptide epitopes. In another embodiment, theheterologous peptide epitopes comprise a CTL peptide epitope and auniversal HTL peptide epitope. In another embodiment, the heterologouspeptide epitopes further comprise one to two or more heterologous CTLpeptide epitopes. In another embodiment, the expression vector comprisesboth HTL and CTL peptide epitopes.

In one embodiment, one of the HTL peptide epitopes is a universal HTLepitope. In another embodiment, the universal HTL epitope is a pan DRepitope. In another embodiment, the pan DR epitope has the sequenceAlaLysPheValAlaAlaTrpThrLeuLysAlaAlaAla (SEQ ID NO:52)

In one embodiment, the peptide epitopes are hepatitis B virus epitopes,hepatitis C virus epitopes, human immunodeficiency virus epitopes, humanpapilloma virus epitopes, MAGE epitopes, PSA epitopes, PSM epitopes, PAPepitopes, p53 epitopes, CEA epitopes, Her2/neu epitopes, or Plasmodiumepitopes. In another embodiment, the peptide epitopes each have asequence selected from the group consisting of the peptides depicted inTables 1-8. In another embodiment, at least one of the peptide epitopesis an analog of a peptide depicted in Tables 1-8.

In one embodiment, the MHC targeting sequence comprises a region of apolypeptide selected from the group consisting of the Ii protein,LAMP-I, HLS-DM, HLA-DO, H2-DO, influenza matrix protein, hepatitis Bsurface antigen, hepatitis B virus core antigen, Ty particle, Ig-αprotein, Ig-β protein, and Ig kappa chain signal sequence.

In one embodiment, the expression vector further comprises a secondpromoter sequence operably linked to a third nucleotide sequenceencoding one or more heterologous HTL or CTL peptide epitopes. Inanother embodiment, the CTL peptide epitope comprises a structural motiffor an HLA supertype, whereby the peptide CTL epitope binds to two ormore members of the supertype with an affinity of greater that 500 nM.In another embodiment, the CTL peptide epitopes have structural motifsthat provide binding affinity for more than one HLA allele supertype.

In one embodiment, the non-human mammal is a transgenic mouse thatexpresses a human HLA allele. In another embodiment, the human HLAallele is selected from the group consisting of AI I and A2. 1. Inanother embodiment, the non-human mammal is a macaque that expresses ahuman HLA allele.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the nucleotide and amino acid sequences (SEQ ID NOS: 1 and2, respectively) of the IiPADRE construct encoding a fusion of themurine Ii gene with a pan DR epitope sequence substituted for the CLIPsequence of the Ii protein.

FIG. 2 shows the nucleotide and amino acid sequences (SEQ ID NOS:3 and4, respectively) of the I80T construct encoding a fusion of thecytoplasmic domain, the transmembrane domain and part of the luminaldomain of the Ii protein fused to multiple MHC class II epitopes.

FIG. 3 shows the nucleotide and amino acid sequences (SEQ ID NOS:5 and6, respectively) of the IiThfull construct encoding a fusion of thecytoplasmic domain, transmembrane domain and a portion of the luminaldomain of the Ii protein fused to multiple T helper epitopes and aminoacid residues 101 to 215 of the Ii protein, which encodes thetrimerization region of the Ii protein.

FIG. 4 shows the nucleotide and amino acid sequences (SEQ ID NOS:7 and8, respectively) of the KappaLAMP-Th construct encoding a fusion of themurine immunoglobulin kappa signal sequence fused to multiple T helperepitopes and the transmembrane and cytoplasmic domains of LAMP-1.

FIG. 5 shows the nucleotide and amino acid sequences (SEQ ID NOS:9 and10, respectively) of the H2M-Th construct encoding a fusion of thesignal sequence of H2-M fused to multiple MHC class II epitopes and thetransmembrane and cytoplasmic domains of H2-M.

FIG. 6 shows the nucleotide and amino acid sequences (SEQ ID NOS:11 and12, respectively) of the H20-Th construct encoding a fusion of thesignal sequence of H2-DO fused to multiple MHC class II epitopes and thetransmembrane and cytoplasmic domains of H2-DO.

FIG. 7 shows the nucleotide and amino acid sequences (SEQ ID NOS:13 and14, respectively) of the PADRE-Influenza matrix construct encoding afusion of a pan DR epitope sequence fused to the amino-terminus ofinfluenza matrix protein.

FIG. 8 shows the nucleotide and amino acid sequences (SEQ ID NOS:15 and16, respectively) of the PADRE-HBV-s construct encoding a fusion of apan DR epitope sequence fused to the amino-terminus of hepatitis B virussurface antigen.

FIG. 9 shows the nucleotide and amino acid sequences (SEQ ID NOS:17 and18, respectively) of the Ig-alphaTh construct encoding a fusion of thesignal sequence of the Ig-a protein fused to multiple MHC class IIepitopes and the transmembrane and cytoplasmic domains of the Ig-αprotein.

FIG. 10 shows the nucleotide and amino acid sequences (SEQ ID NOS:19 and20, respectively) of the Ig-betaTh construct encoding a fusion of thesignal sequence of the Ig-β protein fused to multiple MHC class IIepitopes and the transmembrane and cytoplasmic domains of the Ig-βprotein.

FIG. 11 shows the nucleotide and amino acid sequences (SEQ ID NOS:21 and22, respectively) of the SigTh construct encoding a fusion of the signalsequence of the kappa immunoglobulin fused to multiple MHC class IIepitopes.

FIG. 12 shows the nucleotide and amino acid sequences (SEQ ID NOS:23 and24, respectively) of human HLA-DR, the invariant chain (Ii) protein.

FIG. 13 shows the nucleotide and amino acid sequences (SEQ ID NOS:25 and26, respectively) of human lysosomal membrane glycoprotein-1 (LAMP-1).

FIG. 14 shows the nucleotide and amino acid sequences (SEQ ID NOS:27 and28, respectively) of human HLA-DMB.

FIG. 15 shows the nucleotide and amino acid sequences (SEQ ID NOS:29 and30, respectively) of human HLA-DO beta.

FIG. 16 shows the nucleotide and amino acid sequences (SEQ ID NOS:31 and32, respectively) of the human MB-1 Ig-α.

FIG. 17 shows the nucleotide and amino acid sequences (SEQ ID NOS:33 and34, respectively) of human Ig-β protein.

FIG. 18 shows a schematic diagram depicting the method of generatingsome of the constructs encoding a MHC class II targeting sequence fusedto multiple MHC class II epitopes.

FIG. 19 shows the nucleotide sequence of the vector pEP2 (SEQ ID NO:35).

FIG. 20 shows the nucleotide and amino acid sequences of the vectorpMIN.0 (SEQ ID NO:36 and 37, respectively).

FIG. 21 shows the nucleotide and amino acid sequences of the vectorpMIN.0 (SEQ ID NO:38 and 39, respectively).

FIG. 22. Representative CTL responses in HLA-A2.1/K^(b)-H-2^(bxs) miceimmunized with pMin.1 DNA. Splenocytes from primed animals were culturedin triplicate flasks and stimulated twice in vitro with each peptideepitope. Cytotoxicity of each culture was assayed in a ⁵¹Cr releaseassay against Jurkat-A2.1/K^(b) target cells in the presence (filledsymbols, solid lines) or absence (open symbols, dotted lines) ofpeptide. Each symbol represents the response of a single culture.

FIG. 23. Presentation of viral epitopes to specific CTLs byJurkat-A2.1K^(b) tumor cells transfected with DNA minigene. Twoconstructs were used for transfection, pMin.1 and pMin.2-GFP.pMin.2-GFP-transfected targets cells were sorted by FACS and thepopulation used in this experiment contained 60% fluorescent cells. CTLstimulation was measured by quantitating the amount of IFN-γ release (A,B) or by lysis of ⁵¹Cr-labeled target cells (C, D, hatched bars). CTLswere stimulated with transfected cells (A, C) or with parentalJurkat-A2.1/K^(b) cells in the presence of 1 μg/ml peptide (B, D).Levels of IFN-γ release and cytotoxicity for the different CTL lines inthe absence of epitope ranged from 72-126 pg/ml and 2-6% respectively.

FIG. 24. Summary of modified minigene constructs used to addressvariables critical for in vivo immunogenicity. The followingmodifications were incorporated into the prototype pMin. 1 construct; A,deletion of PADRE HTL epitope; B, incorporation of the native HBV Pol551 epitope that contains an alanine in position 9; C, deletion of theIg kappa signal sequence; and D, switching position of the HBV Env 335and HBV Pol 455 epitopes.

FIG. 25. Examination of variables that may influence pMin. 1immunogenicity. In vivo CTL-inducing activity of pMin.1 is compared tomodified constructs. For ease of comparison, the CTL response induced byeach of the modified DNA minigene constructs (shaded bars) is comparedseparately in each of the four panels to the response induced by theprototype pMin.1 construct (solid bars). The geometric mean response ofCTL-positive cultures from two to five independent experiments areshown. Numbers shown with each bar indicate the number of positivecultures/total number tested for that particular epitope. The ratio ofpositive cultures/total tested for the pMin. 1 group is shown in panel Aand is the same for the remaining Figure panels (see Example V,Materials and Methods, in vitro CTL cultures, for the definition of apositive CTL culture). Theradigm responses were obtained by immunizinganimals with the lipopeptide and stimulating and testing splenocytecultures with the HBV Core 18-27 peptide.

DEFINITIONS

An “HTL” peptide epitopeor an “MHC II epitope” is an MHC class IIrestricted epitope, i.e., one that is bound by an MHC class II molecule.

A “CTL” peptide epitope or an “MHC I epitope” is an MHC class Irestricted epitope, i.e., one that is bound by an MHC class I molecule.

An “MHC targeting sequence” refers to a peptide sequence that targets apolypeptide, e.g., comprising a peptide epitope, to a cytosolic pathway(e.g., an MHC class I antigen processing pathway), en endoplasmicreticulum pathwasy, or an endocytic pathway (e.g., an MHC class IIantigen processing pathway).

The term “heterologous” when used with reference to portions of anucleic acid indicates that the nucleic acid comprises two or moresubsequences that are not found in the same relationship to each otherin nature. For instance, the nucleic acid is typically recombinantlyproduced, having two or more sequences from unrelated genes arranged tomake a new functional nucleic acid, e.g., a promoter from one source anda coding region from another source. Similarly, a heterologous proteinindicates that the protein comprises two or more subsequences that arenot found in the same relationship to each other in nature, e.g., afusion polypeptide comprising subsequence from different polypeptides,peptide epitopes from the same polypeptide that are not naturally in anadjacent position, or repeats of a single peptide epitope.

As used herein, the term “universal MHC class II epitope” or a“universal HTL epitope” refers to a MHC class II peptide epitope thatbinds to gene products of multiple MHC class II alleles. For example,the DR, DP and DQ alleles are human MHC II alleles. Generally, a uniqueset of peptides binds to a particular gene product of a MHC class IIallele. In contrast, a universal MHC class II epitope is able to bind togene products of multiple MHC class II alleles. A universal MHC class IIepitope binds to 2 or more MHC class II alleles, generally 3 or more MHCclass II alleles, and particularly 5 or more MHC class II alleles. Thus,the presence of a universal MHC class II epitope in an expression vectoris advantageous in that it functions to increase the number of allelicMHC class II molecules that can bind to the peptide and, consequently,the number of Helper T lymphocytes that are activated.

Universal MHC class II epitopes are well known in the art and include,for example, epitopes such as the “pan DR epitopes,” also referred to as“PADRE” (Alexander et al., Immunity 1:751-761 (1994); WO 95/07707, U.S.Ser. No. 60/036,713, U.S. Ser. No. 60/037,432, PCT/US98/01373,09/009,953, and U.S. Ser. No. 60/087,192 each of which is incorporatedherein by reference). A “pan DR binding peptide” or a “PADRE” peptide ofthe invention is a peptide capable of binding at least about 7 differentDR molecules, preferably 7 of the 12 most common DR molecules, mostpreferably 9 of the 12 most common DR molecules (DR1, 2w2b, 2w2a, 3,4w4, 4w14, 5, 7, 52a, 52b, 52c, and 53), alternatively, 50% of a panelof DR molecules representative of greater than or equal to 75% of thehuman population, preferably greater than or equal to 80% of the humanpopulation. Pan DR epitopes can bind to a number of DR alleles and arestrongly immunogenic for T cells. For example, pan DR epitopes werefound to be more effective at inducing an immune response than naturalMHC class II epitopes (Alexander, supra). An example of a PADRE epitopeis the peptide AlaLysPheValAlaAlaTrpThrLeuLysAlaAlaAla (SEQ ID NO:52)

With regard to a particular amino acid sequence, an “epitope” is a setof amino acid residues which is involved in recognition by a particularimmunoglobulin, or in the context of T cells, those residues necessaryfor recognition by T cell receptor proteins and/or MajorHistocompatibility Complex (MHC) receptors. In an immune system setting,in vivo or in vitro, an epitope is the collective features of amolecule, such as primary, secondary and tertiary peptide structure, andcharge, that together form a site recognized by an immunoglobulin, Tcell receptor or HLA molecule. Throughout this disclosure epitope andpeptide are often used interchangeably. It is to be appreciated,however, that isolated or purified protein or peptide molecules largerthan and comprising an epitope of the invention are still within thebounds of the invention.

As used herein, “high affinity” with respect to HLA class I molecules isdefined as binding with an IC50 (or K_(D)) of less than 50 nM.“Intermediate affinity” is binding with an IC50 (or K_(D)) of betweenabout 50 and about 500 nM. “High affinity” with respect to binding toHLA class II molecules is defined as binding with an K_(D) of less than100 nM. “Intermediate affinity” is binding with a K_(D) of between about100 and about 1000 nM. Assays for determining binding are described indetail, e.g., in PCT publications WO 94/20127 and WO 94/03205.Alternatively, binding is expressed relative to a reference peptide. Asa particular assay becomes more, or less, sensitive, the IC50s of thepeptides tested may change somewhat. However, the binding relative tothe reference peptide will not significantly change. For example, in anassay run under conditions such that the IC50 of the reference peptideincreases 10-fold, the IC50 values of the test peptides will also shiftapproximately 10-fold. Therefore, to avoid ambiguities, the assessmentof whether a peptide is a good, intermediate, weak, or negative binderis generally based on its IC50, relative to the IC50 of a standardpeptide. Throughout this disclosure, results are expressed in terms of“IC50s.” IC50 is the concentration of peptide in a binding assay atwhich 50% inhibition of binding of a reference peptide is observed.Given the conditions in which the assays are run (i.e., limiting HLAproteins and labeled peptide concentrations), these values approximateKD values. It should be noted that IC50 values can change, oftendramatically, if the assay conditions are varied, and depending on theparticular reagents used (e.g., HLA preparation, etc.). For example,excessive concentrations of HLA molecules will increase the apparentmeasured IC50 of a given ligand.

The terms “identical” or percent “identity,” in the context of two ormore peptide sequences, refer to two or more sequences or subsequencesthat are the same or have a specified percentage of amino acid residuesthat are the same, when compared and aligned for maximum correspondenceover a comparison window, as measured using a sequence comparisonalgorithms using default program parameters or by manual alignment andvisual inspection.

The phrases “isolated” or “biologically pure” refer to material which issubstantially or essentially free from components which normallyaccompany the material as it is found in its native state. Thus,isolated peptides in accordance with the invention preferably do notcontain materials normally associated with the peptides in their in situenvironment.

“Major histocompatibility complex” or “MHC” is a cluster of genes thatplays a role in control of the cellular interactions responsible forphysiologic immune responses. In humans, the MHC complex is also knownas the HLA complex. For a detailed description of the MHC and HLAcomplexes, see Paul, Fundamental Immunology (3rd ed. 1993).

“Human leukocyte antigen” or “HLA” is a human class I or class II majorhistocompatibility complex (MHC) protein (see, e.g., Stites, et al.,Immunology, (8th ed., 1994).

An “HLA supertype or family”, as used herein, describes sets of HLAmolecules grouped on the basis of shared peptide-binding specificities.HLA class I molecules that share somewhat similar binding affinity forpeptides bearing certain amino acid motifs are grouped into HLAsupertypes. The terms HLA superfamily, HLA supertype family, HLA family,and HLA xx-like supertype molecules (where xx denotes a particular HLAtype), are synonyms.

The term “motif” refers to the pattern of residues in a peptide ofdefined length, usually a peptide of from about 8 to about 13 aminoacids for a class I HLA motif and from about 6 to about 25 amino acidsfor a class II HLA motif, which is recognized by a particular HLAmolecule. Peptide motifs are typically different for each proteinencoded by each human HLA allele and differ in the pattern of theprimary and secondary anchor residues.

A “supermotif” is a peptide binding specificity shared by HLA moleculesencoded by two or more HLA alleles. Thus, a preferably is recognizedwith high or intermediate affinity (as defined herein) by two or moreHLA antigens.

“Cross-reactive binding” indicates that a peptide is bound by more thanone HLA molecule; a synonym is degenerate binding.

The term “peptide” is used interchangeably with “oligopeptide” in thepresent specification to designate a series of residues, typicallyL-amino acids, connected one to the other, typically by peptide bondsbetween the α-amino and carboxyl groups of adjacent amino acids. Thepreferred CTL-inducing oligopeptides of the invention are 13 residues orless in length and usually consist of between about 8 and about 11residues, preferably 9 or 10 residues. The preferred HTL-inducingoligopeptides are less than about 50 residues in length and usuallyconsist of between about 6 and about 30 residues, more usually betweenabout 12 and 25, and often between about 15 and 20 residues.

An “immunogenic peptide” or “peptide epitope” is a peptide whichcomprises an allele-specific motif or supermotif such that the peptidewill bind an HLA molecule and induce a CTL and/or HTL response. Thus,immunogenic peptides of the invention are capable of binding to anappropriate HLA molecule and thereafter inducing a cytotoxic T cellresponse, or a helper T cell response, to the antigen from which theimmunogenic peptide is derived.

A “protective immune response” refers to a CTL and/or an HTL response toan antigen derived from an infectious agent or a tumor antigen, whichprevents or at least partially arrests disease symptoms or progression.The immune response may also include an antibody response which has beenfacilitated by the stimulation of helper T cells.

The term “residue” refers to an amino acid or amino acid mimeticincorporated into an oligopeptide by an amide bond or amide bondmimetic.

“Synthetic peptide” refers to a peptide that is not naturally occurring,but is man-made using such methods as chemical synthesis or recombinantDNA technology.

The nomenclature used to describe peptide compounds follows theconventional practice wherein the amino group is presented to the left(the N-terminus) and the carboxyl group to the right (the C-terminus) ofeach amino acid residue. When amino acid residue positions are referredto in a peptide epitope they are numbered in an amino to carboxyldirection with position one being the position closest to the aminoterminal end of the epitope, or the peptide or protein of which it maybe a part. In the formulae representing selected specific embodiments ofthe present invention, the amino- and carboxyl-terminal groups, althoughnot specifically shown, are in the form they would assume at physiologicpH values, unless otherwise specified. In the amino acid structureformulae, each residue is generally represented by standard three letteror single letter designations. The L-form of an amino acid residue isrepresented by a capital single letter or a capital first letter of athree-letter symbol, and the D-form for those amino acids having D-formsis represented by a lower case single letter or a lower case threeletter symbol. Glycine has no asymmetric carbon atom and is simplyreferred to as “Gly” or G.

As used herein, the term “expression vector” is intended to refer to anucleic acid molecule capable of expressing an antigen of interest suchas a MHC class I or class II epitope in an appropriate target cell. Anexpression vector can be, for example, a plasmid or virus, including DNAor RNA viruses. The expression vector contains such a promoter elementto express an antigen of interest in the appropriate cell or tissue inorder to stimulate a desired immune response.

DETAILED DESCRIPTION OF THE INVENTION

Cytotoxic T lymphocytes (CTLs) and helper T lymphocytes (HTLs) arecritical for immunity against infectious pathogens; such as viruses,bacteria, and protozoa; tumor cells; autoimmunne diseases and the like.The present invention provides minigenes that encode peptide epitopeswhich induce a CTL and/or HTL response. The minigenes of the inventionalso include an MHC targeting sequence. A variety of minigenes encodingdifferent epitopes can be tested for immunogenicity using an HLAtransgenic mouse. The epitopes are typically a combination of at leasttwo or more HTL epitopes, or a CTL epitope plus a universal HTL epitope,and optinally include additional HTl and/or CTL epitopes. Two, three,four, five, six, seven, eight, nine, ten, twenty, thirty, forty or aboutfifty different epitopes, either HTL and/or CTL, can be included in theminigene, along with the MHC targeting sequence. The epitopes can havedifferent HLA restriction. Epitopes to be tested include those derivedfrom viruses such as HIV, HBV, HCV, HSV, CMV, HPV, and HTLV; cancerantigens such as p53, Her2/Neu, MAGE, PSA, human papilloma virus, andCEA; parasites such as Trypanosoma, Plasmodium, Leishmania, Giardia,Entamoeba; autoimmune diseases such as rheumatoid arthritis, myestheniagravis, and lupus erythematosus; fungi such as Aspergillus and Candida;and bacteria such as Escherichia coli, Staphylococci, Chlamydia,Mycobacteria, Streptococci, and Pseudomonas. The epitopes to be encodedby the minigene are selected and tested using the methods described inpublished PCT applications WO 93/07421, WO 94/02353, WO 95/01000, WO97/04451, and WO 97/05348, herein incorporated by reference.

HTL and CTL Epitopes

The expression vectors of the invention encode one or more MHC class IIand/or class I epitopes and an MHC targeting sequence. Multiple MHCclass II or class I epitopes present in an expression vector can bederived from the same antigen, or the MHC epitopes can be derived fromdifferent antigens. For example, an expression vector can contain one ormore MHC epitopes that can be derived from two different antigens of thesame virus or from two different antigens of different viruses.Furthermore, any MHC epitope can be used in the expression vectors ofthe invention. For example, any single MHC epitope or a combination ofthe MHC epitopes shown in Tables 1 to 8 can be used in the expressionvectors of the invention. Other peptide epitopes can be selected by oneof skill in the art, e.g., by using a computer to select epitopes thatcontain HLA allele-specific motifs or supermotifs. The expressionvectors of the invention can also encode one or more universal MHC classII epitopes, e.g., PADRE (see, e.g., SEQ ID NO:52

Universal MHC class II epitopes can be advantageously combined withother MHC class I and class II epitopes to increase the number of cellsthat are activated in response to a given antigen and provide broaderpopulation coverage of MHC-reactive alleles. Thus, the expressionvectors of the invention can encode MHC epitopes specific for anantigen, universal MHC class II epitopes, or a combination of specificMHC epitopes and at least one universal MHC class II epitope.

MHC class I epitopes are generally about 5 to 15 amino acids in length,in particular about 8 to 11 amino acids in length. MHC class II epitopesare generally about 10 to 25 amino acids in length, in particular about13 to 21 amino acids in length. A MHC class I or II epitope can bederived from any desired antigen of interest. The antigen of interestcan be a viral antigen, surface receptor, tumor antigen, oncogene,enzyme, or any pathogen, cell or molecule for which an immune responseis desired. Epitopes can be selected based on their ability to bind oneor multiple HLA alleles, and can also be selected using the “analog”technique described below.

Targeting Sequences

The expression vectors of the invention encode one or more MHC epitopesoperably linked to a MHC targeting sequence. The use of a MHC targetingsequence enhances the immune response to an antigen, relative todelivery of antigen alone, by directing the peptide epitope to the siteof MHC molecule assembly and transport to the cell surface, therebyproviding an increased number of MHC molecule-peptide epitope complexesavailable for binding to and activation of T cells.

MHC class I targeting sequences are used in the present invention, e.g.,those sequences that target an MHC class I epitope peptide to acytosolic pathway or to the endoplasmic reticulum (see, e.g., Rammenseeet al., Immunogenetics 41:178-228 (1995)). For example, the cytosolicpathway processes endogenous antigens that are expressed inside thecell. Although not wishing to be bound by any particular theory,cytosolic proteins are thought to be at least partially degraded by anendopeptidase activity of a proteasome and then transported to theendoplasmic reticulum by the TAP molecule (transporter associated withprocessing). In the endoplasmic reticulum, the antigen binds to MHCclass I molecules. Endoplasmic reticulum signal sequences bypass thecytosolic processing pathway and directly target endogenous antigens tothe endoplasmic reticulum, where proteolytic degradation into peptidefragments occurs. Such MHC class I targeting sequences are well known inthe art, and include, e.g., signal sequences such as those from Ig kappa,tissue plasminogen activator or insulin. A preferred signal peptide isthe human Ig kappa chain sequence. Endoplasmic reticulum signalsequences can also be used to target MHC class II epitopes to theendoplasmic reticulum, the site of MHC class I molecule assembly.

MHC class II targeting sequences are also used in the invention, e.g.,those that target a peptide to the endocytic pathway. These targetingsequences typically direct extracellular antigens to enter the endocyticpathway, which results in the antigen being transferred to the lysosomalcompartment where the antigen is proteolytically cleaved into antigenpeptides for binding to MHC class II molecules. As with the normalprocessing of exogenous antigen, a sequence that directs a MHC class IIepitope to the endosomes of the endocytic pathway and/or subsequently tolysosomes, where the MHC class II epitope can bind to a MHC class IImolecule, is a MHC class II targeting sequence. For example, group ofMHC class II targeting sequences useful in the invention are lysosomaltargeting sequences, which localize polypeptides to lysosomes. Since MHCclass II molecules typically bind to antigen peptides derived fromproteolytic processing of endocytosed antigens in lysosomes, a lysosomaltargeting sequence can function as a MHC class II targeting sequence.Lysosomal targeting sequences are well known in the art and includesequences found in the lysosomal proteins LAMP-1 and LAMP-2 as describedby August et al. (U.S. Pat. No. 5,633,234, issued May 27, 1997), whichis incorporated herein by reference.

Other lysosomal proteins that contain lysosomal targeting sequencesinclude HLA-DM. HLA-DM is an endosomal/lysosomal protein that functionsin facilitating binding of antigen peptides to MHC class II molecules.Since it is located in the lysosome, HLA-DM has a lysosomal targetingsequence that can function as a MHC class II molecule targeting sequence(Copier et al., J. Immunol. 157:1017-1027 (1996), which is incorporatedherein by reference).

The resident lysosomal protein HLA-DO can also function as a lysosomaltargeting sequence. In contrast to the above described residentlysosomal proteins LAMP-1 and HLA-DM, which encode specificTyr-containing motifs that target proteins to lysosomes, HLA-DO istargeted to lysosomes by association with HLA-DM (Liljedahl et al., EMBOJ. 15:4817-4824 (1996)), which is incorporated herein by reference.Therefore, the sequences of HLA-DO that cause association with HLA-DMand, consequently, translocation of HLA-DO to lysosomes can be used asMHC class II targeting sequences. Similarly, the murine homolog ofHLA-DO, H2-DO, can be used to derive a MHC class II targeting sequence.A MHC class II epitope can be fused to HLA-DO or H2-DO and targeted tolysosomes.

In another example, the cytoplasmic domains of B cell receptor subunitsIg-α and Ig-β mediate antigen internalization and increase theefficiency of antigen presentation (Bonnerot et al., Immunity 3:335-347(1995)), which is incorporated herein by reference. Therefore, thecytoplasmic domains of the Ig-α and Ig-β proteins can function as MHCclass II targeting sequences that target a MHC class II epitope to theendocytic pathway for processing and binding to MHC class II molecules.

Another example of a MHC class II targeting sequence that directs MHCclass II epitopes to the endocytic pathway is a sequence that directspolypeptides to be secreted, where the polypeptide can enter theendosomal pathway. These MHC class II targeting sequences that directpolypeptides to be secreted mimic the normal pathway by which exogenous,extracellular antigens are processed into peptides that bind to MHCclass II molecules. Any signal sequence that functions to direct apolypeptide through the endoplasmic reticulum and ultimately to besecreted can function as a MHC class II targeting sequence so long asthe secreted polypeptide can enter the endosomal/lysosomal pathway andbe cleaved into peptides that can bind to MHC class II molecules. Anexample of such a fusion is shown in FIG. 11, where the signal sequenceof kappa immunoglobulin is fused to multiple MHC class II epitopes.

In another example, the Ii protein binds to MHC class II molecules inthe endoplasmic reticulum, where it functions to prevent peptidespresent in the endoplasmic reticulum from binding to the MHC class IImolecules. Therefore, fusion of a MHC class II epitope to the Ii proteintargets the MHC class II epitope to the endoplasmic reticulum and a MHCclass II molecule. For example, the CLIP sequence of the Ii protein canbe removed and replaced with a MHC class II epitope sequence so that theMHC class II epitope is directed to the endoplasmic reticulum, where theepitope binds to a MHC class II molecule.

In some cases, antigens themselves can serve as MHC class II or Itargeting sequences and can be fused to a universal MHC class II epitopeto stimulate an immune response. Although cytoplasmic viral antigens aregenerally processed and presented as complexes with MHC class Imolecules, long-lived cytoplasmic proteins such as the influenza matrixprotein can enter the MHC class II molecule processing pathway (Guéguen& Long, Proc. Natl. Acad. Sci. USA 93:14692-14697 (1996)), which isincorporated herein by reference. Therefore, long-lived cytoplasmicproteins can function as a MHC class II targeting sequence. For example,an expression vector encoding influenza matrix protein fused to auniversal MHC class II epitope can be advantageously used to targetinfluenza antigen and the universal MHC class II epitope to the MHCclass II pathway for stimulating an immune response to influenza.

Other examples of antigens functioning as MHC class II targetingsequences include polypeptides that spontaneously form particles. Thepolypeptides are secreted from the cell that produces them andspontaneously form particles, which are taken up into anantigen-presenting cell by endocytosis such as receptor-mediatedendocytosis or are engulfed by phagocytosis. The particles areproteolytically cleaved into antigen peptides after entering theendosomal/lysosomal pathway.

One such polypeptide that spontaneously forms particles is HBV surfaceantigen (HBV-S) (Diminsky et al., Vaccine 15:637-647 (1997); Le Borgneet al., Virology 240:304-315 (1998)), each of which is incorporatedherein by reference. Another polypeptide that spontaneously formsparticles is HBV core antigen (Kuhröber et al., International Immunol.9:1203-1212 (1997)), which is incorporated herein by reference. Stillanother polypeptide that spontaneously forms particles is the yeast Typrotein (Weber et al., Vaccine 13:831-834 (1995)), which is incorporatedherein by reference. For example, an expression vector containing HBV-Santigen fused to a universal MHC class II epitope can be advantageouslyused to target HBV-S antigen and the universal MHC class II epitope tothe MHC class II pathway for stimulating an immune response to HBV.

Binding Affinity of Peptide Epitopes for HLA Molecules

The large degree of HLA polymorphism is an important factor to be takeninto account with the epitope-based approach to vaccine development. Toaddress this factor, epitope selection encompassing identification ofpeptides capable of binding at high or intermediate affinity to multipleHLA molecules is preferably utilized, most preferably these epitopesbind at high or intermediate affinity to two or more allele specific HLAmolecules.

CTL-inducing peptides of interest for vaccine compositions preferablyinclude those that have a binding affinity for class I HLA molecules ofless than 500 nM. HTL-inducing peptides preferably include those thathave a binding affinity for class II HLA molecules of less than 1000 nM.For example, peptide binding is assessed by testing the capacity of acandidate peptide to bind to a purified HLA molecule in vitro. Peptidesexhibiting high or intermediate affinity are then considered for furtheranalysis. Selected peptides are tested on other members of the supertypefamily. In preferred embodiments, peptides that exhibit cross-reactivebinding are then used in vaccines or in cellular screening analyses.

Higher HLA binding affinity is typically correlated with greaterimmunogenicity. Greater immunogenicity can be manifested in severaldifferent ways. Immunogenicity corresponds to whether an immune responseis elicited at all, and to the vigor of any particular response, as wellas to the extent of a population in which a response is elicited. Forexample, a peptide might elicit an immune response in a diverse array ofthe population, yet in no instance produce a vigorous response. Inaccordance with these principles, close to 90% of high binding peptideshave been found to be immunogenic, as contrasted with about 50% of thepeptides which bind with intermediate affinity. Moreover, higher bindingaffinity peptides leads to more vigorous immunogenic responses. As aresult, less peptide is required to elicit a similar biological effectif a high affinity binding peptide is used. Thus, in preferredembodiments of the invention, high binding epitopes are particularlyuseful.

The relationship between binding affinity for HLA class I molecules andimmunogenicity of discrete peptide epitopes on bound antigens has beendetermined for the first time in the art by the present inventors. Thecorrelation between binding affinity and immunogenicity was analyzed intwo different experimental approaches (Sette el al., J. Immunol.153:5586-5592 (1994)). In the first approach, the immunogenicity ofpotential epitopes ranging in HLA binding affinity over a 10,000-foldrange was analyzed in HLA-A*0201 transgenic mice. In the secondapproach, the antigenicity of approximately 100 different hepatitis Bvirus (HBV)-derived potential epitopes, all carrying A*0201 bindingmotifs, was assessed by using PBL (peripheral blood lymphocytes) fromacute hepatitis patients. Pursuant to these approaches, it wasdetermined that an affinity threshold of approximately 500 nM(preferably 50 nM or less) determines the capacity of a peptide epitopeto elicit a CTL response. These data are true for class I bindingaffinity measurements for naturally processed peptides and forsynthesized T cell epitopes. These data also indicate the important roleof determinant selection in the shaping of T cell responses (see, e.g.,Schaeffer et al. Proc. Natl. Acad. Sci. USA 86:4649-4653, 1989).

An affinity threshold associated with immunogenicity in the context ofHLA class II DR molecules has also been delineated (see, e.g., Southwoodet al. J. Immunology 160:3363-3373 (1998), and U.S. Ser. No. 60/087192,filed May 29, 1998). In order to define a biologically significantthreshold of DR binding affinity, a database of the binding affinitiesof 32 DR-restricted epitopes for their restricting element (i.e., theHLA molecule that binds the motif) was compiled. In approximately halfof the cases (15 of 32 epitopes), DR restriction was associated withhigh binding affinities, i.e. binding affinities of less than 100 nM. Inthe other half of the cases (16 of 32), DR restriction was associatedwith intermediate affinity (binding affinities in the 100-1000 nMrange). In only one of 32 cases was DR restriction associated with anIC50 of 1000 nM or greater. Thus, 1000 nM can be defined as an affinitythreshold associated with immunogenicity in the context of DR molecules.

Peptide Epitope Binding Motifs and Supermotifs

In the past few years evidence has accumulated to demonstrate that alarge fraction of HLA class I and class II molecules can be classifiedinto a relatively few supertypes, each characterized by largelyoverlapping peptide binding repertoires, and consensus structures of themain peptide binding pockets.

For HLA molecule pocket analyses, the residues comprising the B and Fpockets of HLA class I molecules as described in crystallographicstudies were analyzed (Guo et al., Nature 360:364 (1992); Saper et al.,J. Mol. Biol. 219:277 (1991); Madden et al., Cell 75:693 (1993); Parhamet al., Immunol. Rev. 143:141 (1995)). In these analyses, residues 9,45, 63, 66, 67, 70, and 99 were considered to make up the B pocket; andthe B pocket was deemed to determine the specificity for the amino acidresidue in the second position of peptide ligands. Similarly, residues77, 80, 81, and 116 were considered to determine the specificity of theF pocket; the F pocket was deemed to determine the specificity for theC-terminal residue of a peptide ligand bound by the HLA class Imolecule.

Through the study of single amino acid substituted antigen analogs andthe sequencing of endogenously bound, naturally processed peptides,critical residues required for allele-specific binding to HLA moleculeshave been identified. The presence of these residues correlates withbinding affinity for HLA molecules. The identification of motifs and/orsupermotifs that correlate with high and intermediate affinity bindingis an important issue with respect to the identification of immunogenicpeptide epitopes for the inclusion in a vaccine. Kast et al. (J.Immunol. 152:3904-3912 (1994)) have shown that motif-bearing peptidesaccount for 90% of the epitopes that bind to allele-specific HLA class Imolecules. In this study all possible peptides of 9 amino acids inlength and overlapping by eight amino acids (240 peptides), which coverthe entire sequence of the E6 and E7 proteins of human papillomavirustype 16, were evaluated for binding to five allele-specific HLAmolecules that are expressed at high frequency among different ethnicgroups. This unbiased set of peptides allowed an evaluation of thepredictive value of HLA class I motifs. From the set of 240 peptides, 22peptides were identified that bound to an allele-specific HLA moleculeswith high or intermediate affinity. Of these 22 peptides, 20, (i.e.,91%), were motif-bearing. Thus, this study demonstrates the value ofmotifs for the identification of peptide epitopes for inclusion in avaccine: application of motif-based identification techniques eliminatesscreening of 90% of the potential epitopes in a target antigen proteinsequence.

Peptides of the present invention may also include epitopes that bind toMHC class II DR molecules. There is a significant difference betweenclass I and class II HLA molecules. This difference corresponds to thefact that, although a stringent size restriction and motif positionrelative to the binding pocket exists for peptides that bind to class Imolecules, a greater degree of heterogeneity in both size and bindingframe position of the motif, relative to the N and C termini of thepeptide, exists for class II peptide ligands.

This increased heterogeneity of HLA class II peptide ligands is due tothe structure of the binding groove of the HLA class II molecule which,unlike its class I counterpart, is open at both ends. Crystallographicanalysis of HLA class II DRB*0101-peptide complexes showed that theresidues occupying position I and position 6 of peptides complexed withDRB*0101 engage two complementary pockets on the DRBa*0101 molecules,with the P1 position corresponding to the most crucial anchor residueand the deepest hydrophobic pocket (see, e.g., Madden, Ann. Rev.Immunol. 13:587 (1995)). Other studies have also pointed to the P6position as a crucial anchor residue for binding to various other DRmolecules.

Thus, peptides of the present invention are identified by any one ofseveral HLA class I or II -specific amino acid motifs (see, e.g., TablesI-III of U.S. Ser. No. 09/226,775, and 09/239,043, herein incorporatedby reference in their entirety). If the presence of the motifcorresponds to the ability to bind several allele-specific HLA antigensit is referred to as a supernotif. The allele-specific HLA moleculesthat bind to peptides that possess a particular amino acid supermotifare collectively referred to as an HLA “supertype.”

Immune Response-Stimulating Peptide Analogs

In general, CTL and HTL responses are not directed against all possibleepitopes. Rather, they are restricted to a few “immunodominant”determinants (Zinkernagel et al., Adv. Immunol. 27:5159 (1979); Benninket al., J. Exp. Med. 168:1935-1939 (1988); Rawle et al., J. Immunol.146:3977-3984 (1991)). It has been recognized that immunodominance(Benacerraf et al., Science 175:273-279 (1972)) could be explained byeither the ability of a given epitope to selectively bind a particularHLA protein (determinant selection theory) (Vitiello et al., J. Immunol.131:1635 (1983)); Rosenthal et al., Nature 267:156-158 (1977)), or beingselectively recognized by the existing TCR (T cell receptor) specificity(repertoire theory) (Klein, Immunology, The Science of Self on selfDiscrimination, pp. 270-310 (1982)). It has been demonstrated thatadditional factors, mostly linked to processing events, can also play akey role in dictating, beyond strict immunogenicity, which of the manypotential determinants will be presented as immunodominant (Sercarz etal., Annu. Rev. Immunol. 11:729-766 (1993)).

The concept of dominance and subdominance is relevant to immunotherapyof both infectious diseases and cancer. For example, in the course ofchronic viral disease, recruitment of subdominant epitopes can beimportant for successful clearance of the infection, especially ifdominant CTL or HTL specificities have been inactivated by functionaltolerance, suppression, mutation of viruses and other mechanisms (Francoet al., Curr. Opin. Immunol. 7:524-531 (1995)). In the case of cancerand tumor antigens, CTLs recognizing at least some of the highestbinding affinity peptides might be functionally inactivated. Lowerbinding affinity peptides are preferentially recognized at these times,and may therefore be preferred in therapeutic or prophylacticanti-cancer vaccines.

In particular, it has been noted that a significant number of epitopesderived from known non-viral tumor associated antigens (TAA) bind HLAclass I with intermediate affinity (IC50 in the 50-500 nM range). Forexample, it has been found that 8 of 15 known TAA peptides recognized bytumor infiltrating lymphocytes (TIL) or CTL bound in the 50-500 nMrange. (These data are in contrast with estimates that 90% of knownviral antigens were bound by HLA class I molecules with IC50 of 50 nM orless, while only approximately 10% bound in the 50-500 nM range (Setteet al., J. Immunol., 153:558-5592 (1994)). In the cancer setting thisphenomenon is probably due to elimination, or functional inhibition ofthe CTL recognizing several of the highest binding peptides, presumablybecause of T cell tolerization events.

Without intending to be bound by theory, it is believed that because Tcells to dominant epitopes may have been clonally deleted, selectingsubdominant epitopes may allow extant T cells to be recruited, whichwill then lead to a therapeutic or prophylactic response. However, thebinding of HLA molecules to subdominant epitopes is often less vigorousthan to dominant ones. Accordingly, there is a need to be able tomodulate the binding affinity of particular immunogenic epitopes for oneor more HLA molecules, and thereby to modulate the immune responseelicited by the peptide, for example to prepare analog peptides whichelicit a more vigorous response. This ability would greatly enhance theusefulness of peptide-based vaccines and therapeutic agents.

Thus, although peptides with suitable cross-reactivity among all allelesof a superfamily are identified by the screening procedures describedabove, cross-reactivity is not always as complete as possible, and incertain cases procedures to further increase cross-reactivity ofpeptides can be useful; moreover, such procedures can also be used tomodify other properties of the peptides such as binding affinity orpeptide stability. Having established the general rules that governcross-reactivity of peptides for HLA alleles within a given motif orsupermotif, modification (i.e., analoging) of the structure of peptidesof particular interest in order to achieve broader (or otherwisemodified) HLA binding capacity can be performed. More specifically,peptides which exhibit the broadest cross-reactivity patterns, can beproduced in accordance with the teachings herein. The present conceptsrelated to analog generation are set forth in greater detail inco-pending U.S. Ser. No. 09/226,775.

In brief, the strategy employed utilizes the motifs or supermotifs whichcorrelate with binding to certain HLA class I and II molecules. Themotifs or supermotifs are defined by having primary anchors, and in manycases secondary anchors (see Tables I-III of U.S. Ser. No. 09/226,775).Analog peptides can be created by substituting amino acids residues atprimary anchor, secondary anchor, or at primary and secondary anchorpositions. Generally, analogs are made for peptides that already bear amotif or supermotif. Preferred secondary anchor residues of supermotifsand motifs that have been defined for HLA class I and class II bindingpeptides are shown in Tables II and III, respectively, of U.S. Ser. No.09/226,775.

For a number of the motifs or supermotifs in accordance with theinvention, residues are defined which are deleterious to binding toallele-specific HLA molecules or members of HLA supertypes that bind tothe respective motif or supermotif (see Tables II and III of U.S. Ser.No. 09/226,775). Accordingly, removal of such residues that aredetrimental to binding can be performed in accordance with the methodsdescribed therein. For example, in the case of the A3 supertype, whenall peptides that have such deleterious residues are removed from thepopulation of analyzed peptides, the incidence of cross-reactivityincreases from 22% to 37% (I., Sidney et al., Hu. Immunol. 45:79(1996)). Thus, one strategy to improve the cross-reactivity of peptideswithin a given supermotif is simply to delete one or more of thedeleterious residues present within a peptide and substitute a small“neutral” residue such as Ala (that may not influence T cell recognitionof the peptide). An enhanced likelihood of cross-reactivity is expectedif, together with elimination of detrimental residues within a peptide,“preferred” residues associated with high affinity binding to anallele-specific HLA molecule or to multiple HLA molecules within asuperfamily are inserted.

To ensure that an analog peptide, when used as a vaccine, actuallyelicits a CTL response to the native epitope in vivo (or, in the case ofclass II epitopes, a failure to elicit helper T cells that cross-reactwith the wild type peptides), the analog peptide may be used to immunizeT cells in vitro from individuals of the appropriate HLA allele.Thereafter, the immunized cells' capacity to induce lysis of wild typepeptide sensitized target cells is evaluated. In both class I and classII systems it will be desirable to use as targets, cells that have beeneither infected or transfected with the appropriate genes to establishwhether endogenously produced antigen is also recognized by the relevantT cells.

Another embodiment of the invention is to create analogs of weak bindingpeptides, to thereby ensure adequate numbers of cross-reactive cellularbinders. Class I peptides exhibiting binding affinities of 500-50000 nM,and carrying an acceptable but suboptimal primary anchor residue at oneor both positions can be “fixed” by substituting preferred anchorresidues in accordance with the respective supertype. The analogpeptides can then be tested for crossbinding activity.

Another embodiment for generating effective peptide analogs involves thesubstitution of residues that have an adverse impact on peptidestability or solubility in, e.g., a liquid environment. Thissubstitution may occur at any position of the peptide epitope. Forexample, a cysteine (C) can be substituted out in favor of gamma-aminobutyric acid. Due to its chemical nature, cysteine has the propensity toform disulfide bridges and sufficiently alter the peptide structurallyso as to reduce binding capacity. Substituting gamma-amino butyric acidfor C not only alleviates this problem, but actually improves bindingand crossbinding capability in certain instances (Sette et al, In:Persistent Viral Infections (Ahmed & Chen, eds., 1998)). Substitution ofcysteine with gamma-amino butyric acid may occur at any residue of apeptide epitope, i.e., at either anchor or non-anchor positions.

Expression Vectors and Construction of a Minigene

The expression vectors of the invention contain at least one promoterelement that is capable of expressing a transcription unit encoding theantigen of interest, for example, a MHC class I epitope or a MHC classII epitope and an MHC targeting sequence in the appropriate cells of anorganism so that the antigen is expressed and targeted to theappropriate MHC molecule. For example, if the expression vector isadministered to a mammal such as a human, a promoter element thatfunctions in a human cell is incorporated into the expression vector. Anexample of an expression vector useful for expressing the MHC class IIepitopes fused to MHC class II targeting sequences and the MHC class Iepitopes described herein is the pEP2 vector described in Example IV.

This invention relies on routine techniques in the field of recombinantgenetics. Basic texts disclosing the general methods of use in thisinvention include Sambrook et al., Molecular Cloning, A LaboratoryManual (2nd ed. 1989); Kriegler, Gene Transfer and Expression: ALaboratory Manual (1990); and Current Protocols in Molecular Biology(Ausubel et al., eds., 1994); Oligonucleotide Synthesis: A PracticalApproach (Gait, ed., 1984); Kuijpers, Nucleic Acids Research 18(17):5197(1994); Dueholm, J. Org. Chem. 59:5767-5773 (1994); Methods in MolecularBiology, volume (Agrawal, ed.); and Tijssen, Laboratory Techniques inBiochemistry and Molecular Biology—Hybridization with Nucleic AcidProbes, e.g., Part I, chapter 2 “Overview of principles of hybridizationand the strategy of nucleic acid probe assays” (1993)).

The minigenes are comprised of two or many different epitopes (see,e.g., Tables 1-8). The nucleic acid encoding the epitopes are assembledin a minigene according to standard techniques. In general, the nucleicacid sequences encoding minigene epitopes are isolated usingamplification techniques with oligonucleotide primers, or are chemicallysynthesized. Recombinant cloning techniques can also be used whenappropriate. Oligonucleotide sequences are selected which either amplify(when using PCR to assemble the minigene) or encode (when usingsynthetic oligonucleotides to assemble the minigene) the desiredepitopes.

Amplification techniques using primers are typically used to amplify andisolate sequences encoding the epitopes of choice from DNA or RNA (seeU.S. Pat. Nos. 4,683,195 and 4,683,202; PCR Protocols: A Guide toMethods and Applications (Innis et al., eds, 1990)). Methods such aspolymerase chain reaction (PCR) and ligase chain reaction (LCR) can beused to amplify epitope nucleic acid sequences directly from mRNA, fromcDNA, from genomic libraries or cDNA libraries. Restriction endonucleasesites can be incorporated into the primers. Minigenes amplified by thePCR reaction can be purified from agarose gels and cloned into anappropriate vector.

Synthetic oligonucleotides can also be used to construct minigenes. Thismethod is performed using a series of overlapping oligonucleotides,representing both the sense and non-sense strands of the gene. These DNAfragments are then annealed, ligated and cloned. Oligonucleotides thatare not commercially available can be chemically synthesized accordingto the solid phase phosphoramidite triester method first described byBeaucage & Caruthers, Tetrahedron Letts. 22:1859-1862 (1981), using anautomated synthesizer, as described in Van Devanter et. al., NucleicAcids Res. 12:6159-6168 (1984). Purification of oligonucleotides is byeither native acrylamide gel electrophoresis or by anion-exchange HPLCas described in Pearson & Reanier, J. Chrom. 255:137-149 (1983).

The epitopes of the minigene are typically subcloned into an expressionvector that contains a strong promoter to direct transcription, as wellas other regulatory sequences such as enhancers and polyadenylationsites. Suitable promoters are well known in the art and described, e.g.,in Sambrook et al. and Ausubel et al. Eukaryotic expression systems formammalian cells are well known in the art and are commerciallyavailable. Such promoter elements include, for example, cytomegalovirus(CMV), Rous sarcoma virus LTR and SV40.

The expression vector typically contains a transcription unit orexpression cassette that contains all the additional elements requiredfor the expression of the minigene in host cells. A typical expressioncassette thus contains a promoter operably linked to the minigene andsignals required for efficient polyadenylation of the transcript.Additional elements of the cassette may include enhancers and intronswith functional splice donor and acceptor sites.

In addition to a promoter sequence, the expression cassette can alsocontain a transcription termination region downstream of the structuralgene to provide for efficient termination. The termination region may beobtained from the same gene as the promoter sequence or may be obtainedfrom different genes.

The particular expression vector used to transport the geneticinformation into the cell is not particularly critical. Any of theconventional vectors used for expression in eukaryotic cells may beused. Expression vectors containing regulatory elements from eukaryoticviruses are typically used in eukaryotic expression vectors, e.g., SV40vectors, papilloma virus vectors, and vectors derived from Epstein Barvirus. Other exemplary eukaryotic vectors include pMSG, pAV009/A+,pMTO10/A+, pMAMneo-5, baculovirus pDSVE, and any other vector allowingexpression of proteins under the direction of the SV40 early promoter,SV40 later promoter, metallothionein promoter, murine mammary tumorvirus promoter, Rous sarcoma virus promoter, polyhedrin promoter, orother promoters shown effective for expression in eukaryotic cells. Inone embodiment, the vector pEP2 is used in the present invention.

Other elements that are typically included in expression vectors alsoinclude a replicon that functions in E. coli, a gene encoding antibioticresistance to permit selection of bacteria that harbor recombinantplasmids, and unique restriction sites in nonessential regions of theplasmid to allow insertion of eukaryotic sequences. The particularantibiotic resistance gene chosen is not critical, any of the manyresistance genes known in the art are suitable. The prokaryoticsequences are preferably chosen such that they do not interfere with thereplication of the DNA in eukaryotic cells, if necessary.

Administration In Vivo

The invention also provides methods for stimulating an immune responseby administering an expression vector of the invention to an individual.Administration of an expression vector of the invention for stimulatingan immune response is advantageous because the expression vectors of theinvention target MHC epitopes to MHC molecules, thus increasing thenumber of CTL and HTL activated by the antigens encoded by theexpression vector.

Initially, the expression vectors of the invention are screened in mouseto determine the expression vectors having optimal activity instimulating a desired immune response. Initial studies are thereforecarried out, where possible, with mouse genes of the MHC targetingsequences. Methods of determining the activity of the expression vectorsof the invention are well known in the art and include, for example, theuptake of ³H-thymidine to measure T cell activation and the release of⁵¹Cr to measure CTL activity as described below in Examples II and III.Experiments similar to those described in Example IV are performed todetermine the expression vectors having activity at stimulating animmune response. The expression vectors having activity are furthertested in human. To circumvent potential adverse immunological responsesto encoded mouse sequences, the expression vectors having activity aremodified so that the MHC class II targeting sequences are derived fromhuman genes. For example, substitution of the analogous regions of thehuman homologs of genes containing various MHC class II targetingsequences are substituted into the expression vectors of the invention.Examples of such human homologs of genes containing MHC class IItargeting sequences are shown in FIGS. 12 to 17. Expression vectorscontaining human MHC class II targeting sequences, such as thosedescribed in Example I below, are tested for activity at stimulating animmune response in human.

The invention also relates to pharmaceutical compositions comprising apharmaceutically acceptable carrier and an expression vector of theinvention. Pharmaceutically acceptable carriers are well known in theart and include aqueous or non-aqueous solutions, suspensions andemulsions, including physiologically buffered saline, alcohol/aqueoussolutions or other solvents or vehicles such as glycols, glycerol, oilssuch as olive oil or injectable organic esters.

A pharmaceutically acceptable carrier can contain physiologicallyacceptable compounds that act, for example, to stabilize the expressionvector or increase the absorption of the expression vector. Suchphysiologically acceptable compounds include, for example,carbohydrates, such as glucose, sucrose or dextrans, antioxidants suchas ascorbic acid or glutathione, chelating agents, low molecular weightpolypeptides, antimicrobial agents, inert gases or other stabilizers orexcipients. Expression vectors can additionally be complexed with othercomponents such as peptides, polypeptides and carbohydrates. Expressionvectors can also be complexed to particles or beads that can beadministered to an individual, for example, using a vaccine gun. Oneskilled in the art would know that the choice of a pharmaceuticallyacceptable carrier, including a physiologically acceptable compound,depends, for example, on the route of administration of the expressionvector.

The invention further relates to methods of administering apharmaceutical composition comprising an expression vector of theinvention to stimulate an immune response. The expression vectors areadministered by methods well known in the art as described in Donnellyet al. (Ann. Rev. Immunol. 15:617-648 (1997)); Felgner et al. (U.S. Pat.No. 5,580,859, issued Dec. 3, 1996); Felgner (U.S. Pat. No. 5,703,055,issued Dec. 30, 1997); and Carson et al. U.S. Pat. No. 5,679,647, issuedOct. 21, 1997), each of which is incorporated herein by reference. Inone embodiment, the minigene is administered as naked nucleic acid.

A pharmaceutical composition comprising an expression vector of theinvention can be administered to stimulate an immune response in asubject by various routes including, for example, orally,intravaginally, rectally, or parenterally, such as intravenously,intramuscularly, subcutaneously, intraorbitally, intracapsularly,intraperitoneally, intracisternally or by passive or facilitatedabsorption through the skin using, for example, a skin patch ortransdermal iontophoresis, respectively. Furthermore, the compositioncan be administered by injection, intubation or topically, the latter ofwhich can be passive, for example, by direct application of an ointmentor powder, or active, for example, using a nasal spray or inhalant. Anexpression vector also can be administered as a topical spray, in whichcase one component of the composition is an appropriate propellant. Thepharmaceutical composition also can be incorporated, if desired, intoliposomes, microspheres or other polymer matrices (Felgner et al., U.S.Pat. No. 5,703,055; Gregoriadis, Liposome Technology, Vols. I to III(2nd ed. 1993), each of which is incorporated herein by reference).Liposomes, for example, which consist of phospholipids or other lipids,are nontoxic, physiologically acceptable and metabolizable carriers thatare relatively simple to make and administer.

The expression vectors of the invention can be delivered to theinterstitial spaces of tissues of an animal body (Feigner et al., U.S.Pat. Nos. 5,580,859 and 5,703,055). Administration of expression vectorsof the invention to muscle is a particularly effective method ofadministration, including intradermal and subcutaneous injections andtransdermal administration. Transdermal administration, such as byiontophoresis, is also an effective method to deliver expression vectorsof the invention to muscle. Epidermal administration of expressionvectors of the invention can also be employed. Epidermal administrationinvolves mechanically or chemically irritating the outermost layer ofepidermis to stimulate an immune response to the irritant (Carson etal., U.S. Pat. No. 5,679,647).

Other effective methods of administering an expression vector of theinvention to stimulate an immune response include mucosal administration(Carson et al., U.S. Pat. No. 5,679,647). For mucosal administration,the most effective method of administration includes intranasaladministration of an appropriate aerosol containing the expressionvector and a pharmaceutical composition. Suppositories and topicalpreparations are also effective for delivery of expression vectors tomucosal tissues of genital, vaginal and ocular sites. Additionally,expression vectors can be complexed to particles and administered by avaccine gun.

The dosage to be administered is dependent on the method ofadministration and will generally be between about 0.1 μg up to about200 μg. For example, the dosage can be from about 0.05 μg/kg to about 50mg/kg, in particular about 0.005-5 mg/kg. An effective dose can bedetermined, for example, by measuring the immune response afteradministration of an expression vector. For example, the production ofantibodies specific for the MHC class II epitopes or MHC class Iepitopes encoded by the expression vector can be measured by methodswell known in the art, including ELISA or other immunological assays. Inaddition, the activation of T helper cells or a CTL response can bemeasured by methods well known in the art including, for example, theuptake of ³H-thymidine to measure T cell activation and the release of⁵¹Cr to measure CTL activity (see Examples II and III below).

The pharmaceutical compositions comprising an expression vector of theinvention can be administered to mammals, particularly humans, forprophylactic or therapeutic purposes. Examples of diseases that can betreated or prevented using the expression vectors of the inventioninclude infection with HBV, HCV, HIV and CMV as well as prostate cancer,renal carcinoma, cervical carcinoma, lymphoma, condyloma acuminatum andacquired immunodeficiency syndrome (AIDS).

In therapeutic applications, the expression vectors of the invention areadministered to an individual already suffering from cancer, autoimmunedisease or infected with a virus. Those in the incubation phase or acutephase of the disease can be treated with expression vectors of theinvention, including those expressing all universal MHC class IIepitopes, separately or in conjunction with other treatments, asappropriate.

In therapeutic and prophylactic applications, pharmaceuticalcompositions comprising expression vectors of the invention areadministered to a patient in an amount sufficient to elicit an effectiveimmune response to an antigen and to ameliorate the signs or symptoms ofa disease. The amount of expression vector to administer that issufficient to ameliorate the signs or symptoms of a disease is termed atherapeutically effective dose. The amount of expression vectorsufficient to achieve a therapeutically effective dose will depend onthe pharmaceutical composition comprising an expression vector of theinvention, the manner of administration, the state and severity of thedisease being treated, the weight and general state of health of thepatient and the judgment of the prescribing physician.

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be readily apparent to one of ordinary skill inthe art in light of the teachings of this invention that certain changesand modifications may be made thereto without departing from the spiritor scope of the appended claims.

EXAMPLES

The following example is provided by way of illustration only and not byway of limitation. Those of skill in the art will readily recognize avariety of noncritical parameters that could be changed or modified toyield essentially similar results.

Example 1 Construction of Expression Vectors Containing MHC Class IIEpitopes

This example shows construction of expression vectors containing MHCclass II epitopes that can be used to target antigens to MHC class IImolecules.

Expression vectors comprising DNA constructs were prepared usingoverlapping oligonucleotides, polymerase chain reaction (PCR) andstandard molecular biology techniques (Dieffenbach & Dveksler, PCRPrimer: A Laboratory Manual (1995); Sambrook et al., Molecular Cloning:A Laboratory Manual (2nd ed., 1989), each of which is incorporatedherein by reference).

To generate full length wild type Ii, the full length invariant chainwas amplified, cloned, and sequenced and used in the construction of thethree invariant chain constructs. Except where noted, the source of cDNAfor all the constructs listed below was Mouse Spleen Marathon-Ready cDNAmade from Balb/c males (Clontech; Palo Alto Calif.). The primer pairswere the oligonucleotide GCTAGCGCCGCCACCATGGATGACCAACGCGACCTC (SEQ IDNO:40), which is designated murIi-F and contains an NheI site followedby the consensus Kozak sequence and the 5′ end of the Ii cDNA; and theoligonucleotide GGTACCTCACAGGGTGACTTGACCCAG (SEQ ID NO:41), which isdesignated murIi-R and contains a KpnI site and the 3′ end of the Iicoding sequence.

For the PCR reaction, 5 μl of spleen cDNA and 250 nM of each primer werecombined in a 100 μl reaction with 0.25 mM each dNTP and 2.5 units ofPfu polymerase in Pfu polymerase buffer containing 10 mM KCl, 10 mMNM₄)₂SO₄, 20 mM Tris-chloride, pH 8.75, 2 MM MgSO₄, 0.1% TRITON X-100and 100 μg/ml bovine serum albumin (BSA). A Perkin/Elmer 9600 PCRmachine (Perkin Elmer; Foster City Calif.) was used and the cyclingconditions were: 1 cycle of 95° C. for 5 minutes, followed by 30 cyclesof 95° C. for 15 seconds, 52° C. for 30 seconds, and 72° C. for 1minute. The PCR reaction was run on a 1% agarose gel, and the 670 basepair product was cut out, purified by spinning through a MilliporeUltrafree-MC filter (Millipore; Bedford Mass.) and cloned into pCR-Bluntfrom Invitrogen (San Diego, Calif.). Individual clones were screened bysequencing, and a correct clone (named bIi#3) was used as a template forthe helper constructs.

DNA constructs containing pan DR epitope sequences and MHC II targetingsequences derived from the Ii protein were prepared. The Ii murineprotein has been previously described (Zhu & Jones, Nucleic Acids Res.17:447-448 (1989)), which is incorporated herein by reference. Briefly,the IiPADRE construct contains the full length Ii sequence with PADREprecisely replacing the CLIP region. The DNA construct encodes aminoacids 1 through 87 of invariant chain, followed with the 13 amino acidPADRE sequence (SEQ ID NO.52) and the rest of the invariant chain DNAsequence (amino acids 101-215). The construct was amplified in 2overlapping halves that were joined to produce the final construct. Thetwo primers used to amplify the 5′ half were murIi-F and theoligonucleotide CAGGGTCCAGGCAGCCACGAACTTGGCCACAGGTTTGGCAGA (SEQ IDNO:42), which is designated IiPADRE-R. The IiPADRE-R primer includesnucleotides 303-262 of IiPADRE. The 3′ half was amplified with theprimer GGCTGCCTGGACCCTGAAGGCTGCCGCTATGTCCATGGATAAC (SEQ ID NO:43), whichis designated IiPADRE-F and includes nucleotides 288-330 of IiPADRE; andmurIi-R. The PCR conditions were the same as described above, and thetwo halves were isolated by agarose gel electrophoresis as describedabove.

Ten microliters of each PCR product was combined in a 100 μl PCRreaction with an annealing temperature of 50° C. for five cycles togenerate a full length template. Primers murIi-F and murIi-R were addedand 25 more cycles carried out. The full length IiPADRE product wasisolated, cloned, and sequenced as described above. This constructcontains the murine Ii gene with a pan DR epitope sequence substitutedfor the CLIP sequence of Ii (FIG. 1).

A DNA construct, designated I80T, containing the cytoplasmic domain, thetransmembrane domain and part of the luminal domain of Ii fused to astring of multiple MHC class II epitopes was constructed (FIG. 2).Briefly, the string of multiple MHC class II epitopes was constructedwith three overlapping oligonucleotides (oligos). Each oligo overlappedits neighbor by 15 nucleotides and the final MHC class II epitope stringwas assembled by extending the overlapping oligonucleotides in threesets of reactions using PCR. The three oligonucleotides were: oligo 1,nucleotides 241-310,CTTCGCATGAAGCTTATCAGCCAGGCTGTGCACGCCGCTCACGCCGAAATCAA CGAAGCTGGAAGAACCC(SEQ ID NO:44);

oligo 2, nucleotides 364-295,TTCTGGTCAGCAGAAAGAACAGGATAGGAGCGTTTGGAGGGCGATAAGCTGG AGGGGTTCTTCCAGCTTC(SEQ ID NO:45); and

oligo 3, nucleotides 350-42,TTCTGCTGACCAGAATCCTGACAATCCCCCAGTCCCTGGACGCCAAGTTCGTG GCTGCCTGGACCCTGAAG(SEQ ID NO:46).

For the first PCR reaction, 5 μg of oligos 1 and 2 were combined in a100 μl reaction containing Pfu polymerase. A Perkin/Elmer 9600 PCRmachine was used and the annealing temperature used was 45° C. The PCRproduct was gel-purified, and a second reaction containing the PCRproduct of oligos 1 and 2 with oligo 3 was annealed and extended for 10cycles before gel purification of the full length product to be used asa “mega-primer.”

The I80T construct was made by amplifying bIi#3 with murIi-F and themega-primer. The cycling conditions were: 1 cycle of 95° C. for 5minutes, followed by 5 cycles of 95° C. for 15 seconds, 37° C. for 30seconds, and 72° C. for 1 minute. Primer Help-epR was added and anadditional 25 cycles were carried out with the annealing temperatureraised to 47° C. The Help-epR primer GGTACCTCAAGCGGCAGCCTTCAGGGTCCAGGCA(SEQ ID NO:47) corresponds to nucleotides 438-405. The full length I80Tproduct was isolated, cloned, and sequenced as above.

The I80T construct (FIG. 2) encodes amino acid residues 1 through 80 ofIi, containing the cytoplasmic domain, the transmembrane domain and partof the luminal domain, fused to a string of multiple MHC class IIepitopes corresponding to: amino acid residues 323-339 of ovalbumin(IleSerGlnAlaValHisAlaAlaHisAlaGluIleAsnGluAlaGlyArg; SEQ ID NO:48);amino acid residues 128 to 141 of HBV core antigen (amino acidsThrProProAlaTyrArgProProAsnAlaProIleLeu; SEQ ID NO:49); amino acidresidues 182 to 196 of HBV env (amino acidsPhePheLeuLeuThrArglleLeuThrIleProGlnSerLeuAsp; SEQ ID NO:50); and thepan DR sequence designated SEQ ID NO:52)

A DNA construct containing the cytoplasmic domain, transmembrane domainand a portion of the luminal domain of Ii fused to the MHC class IIepitope string shown in FIG. 2 and amino acid residues 101 to 215 of Iiencoding the trimerization region of Ii was generated (FIG. 3). Thisconstruct, designated IiThfull, encodes the first 80 amino acids ofinvariant chain followed by the MHC class II epitope string (replacingCLIP) and the rest of the invariant chain (amino acids 101-215).Briefly, the construct was generated as two overlapping halves that wereannealed and extended by PCR to yield the final product.

The 5′ end of IiThfull was made by amplifying I80T with murIi-F (SEQ IDNO:40) and Th-Pad-R. The Th-Pad-R primer AGCGGCAGCCTTCAGGGTC (SEQ IDNO:51) corresponds to nucleotides 429-411. The 3′ half was made byamplifying bIi#3 with IiPADRE-F and murIi-R (SEQ ID NO:41). TheIiPADRE-F primer GGCTGCCTGGACCCTGAAGGCTGCCGCTATGTCCATGGATAAC (SEQ IDNO:43) corresponds to nucleotides 402-444. Each PCR product was gelpurified and mixed, then denatured, annealed, and extended by fivecycles of PCR. Primers murIi-F (SEQ ID NO:40) and murIi-R (SEQ ID NO:41)were added and another 25 cycles performed. The full length product wasgel purified, cloned, and sequenced.

All of the remaining constructs described below were made essentiallyaccording to the scheme shown in FIG. 18. Briefly, primer pairs 1F plus1R, designated below for each specific construct, were used to amplifythe specific signal sequence and contained an overlapping 15 base pairtail identical to the 5′ end of the MHC class II epitope string. Primerpair Th-ova-F, ATCAGCCAGGCTGTGCACGC (SEQ ID NO:53), plus Th-Pad-R (SEQID NO:51) were used to amplify the MHC class II epitope string. A 15base pair overlap and the specific transmembrane and cytoplasmic tailcontaining the targeting signals were amplified with primer pairs 2Fplus 2R.

All three pieces of each cDNA were amplified using the followingconditions: 1 cycle of 95° C. for 5 minutes, followed by 30 cycles of95° C. for 15 seconds, 52° C. for 30 seconds, and 72° C. for 1 minute.Each of the three fragments was agrose-gel purified, and the signalsequence and MHC class II string fragments were combined and joined byfive cycles in a second PCR. After five cycles, primers 1F and Th-Pad-Rwere added for 25 additional cycles and the PCR product was gelpurified. This signal sequence plus MHC class II epitope string fragmentwas combined with the transmembrane plus cytoplasmic tail fragment forthe final PCR. After five cycles, primers 1F plus 2R were added for 25additional cycles and the product was gel purified, cloned andsequenced.

A DNA construct containing the murine immunoglobulin kappa signalsequence fused to the T helper epitope string shown in FIG. 2 and thetransmembrane and cytoplasmic domains of LAMP-1 was generated (FIG. 4)(Granger et al., J. Biol. Chem. 265:12036-12043 (1990)), which isincorporated by reference (mouse LAMP-1 GenBank accession No. M32015).This construct, designated kappaLAMP-Th, contains the consensus mouseimmunoglobulin kappa signal sequence and was amplified from a plasmidcontaining full length immunoglobulin kappa as depicted in FIG. 18. Theprimer 1F used was the oligonucleotide designated KappaSig-F,GCTAGCGCCGCCACCATGGGAATGCAG (SEQ ID NO:54).

The primer 1R used was the oligonucleotide designated Kappa-Th-R,CACAGCCTGGCTGATTCCTCTGGACCC (SEQ ID NO:55).

The primer 2F used was the oligonucleotide designated PAD/LAMP-F,CTGAAGGCTGCCGCTAACAACATGTTGATCCCC (SEQ ID NO:56). The primer 2R used wasthe oligonucleotide designated LAMP-CYTOR, GGTACCCTAGATGGTCTGATAGCC (SEQID NO:57).

A DNA construct containing the signal sequence of H2-M fused to the MHCclass II epitope string shown in FIG. 2 and the transmembrane andcytoplasmic domains of H2-M was generated (FIG. 5). The mouse H2-M genehas been described previously, Peleraux et al., Immunogenetics43:204-214 (1996)), which is incorporated herein by reference. Thisconstruct was designated H2M-Th and was constructed as depicted in FIG.18. The primer 1F used was the oligonucleotide designated H2-Mb-1F, GCCGCT AGC GCC GCC ACC ATG GCT GCA CTC TGG (SEQ ID NO:58). The primer 1Rused was the oligonucleotide designated H2-Mb-1R, CAC AGC CTG GCT GATCCC CAT ACA GTG CAG (SEQ ID NO:59). The primer 2F used was theoligonucleotide designated H2-Mb-2F, CTG AAG GCT GCC GCT AAG GTC TCT GTGTCT (SEQ ID NO:60). The primer 2R used was the oligonucleotidedesignated H2-Mb-2R, GCG GGTACC CTAATG CCG TCC TTC (SEQ ID NO:61).

A DNA construct containing the signal sequence of H2-DO fused to the MHCclass II epitope string shown in FIG. 2 and the transmembrane andcytoplasmic domains of H2-DO was generated (FIG. 6). The mouse H2-DOgene has been described previously (Larhammar et al., J. Biol. Chem.260:14111-14119 (1985)), which is incorporated herein by reference(GenBank accession No. M19423). This construct, designated HBO-Th, wasconstructed as depicted in FIG. 18. The primer 1F used was theoligonucleotide designated H2-Ob-1F, GCG GCT AGC GCC GCC ACC ATG GGC GCTGGG AGG (SEQ ID NO:62). The primer 1R used was the oligonucleotidedesignated H2-Ob-1R, TGC ACA GCC TGG CTG ATG GAA TCC AGC CTC (SEQ IDNO:63). The primer 2F used was the oligonucleotide designated H2-Ob-2F,CTG AAG GCT GCC GCT ATA CTG AGT GGA GCT (SEQ ID NO:64). The primer 2Rused was the oligonucleotide designated H2-Ob-2R, GCC GGT ACC TCA TGTGAC ATG TCC CG (SEQ ID NO:65).

A DNA construct containing a pan DR epitope sequence (SEQ ID NO:52)fused to the amino-terminus of influenza matrix protein is generated(FIG. 7). This construct, designated PADRE-Influenza matrix, containsthe universal MHC class II epitope PADRE attached to the amino terminusof the influenza matrix coding sequence. The construct is made using along primer on the 5′ end primer. The 5′ primer is the oligonucleotideGCTAGCGCCGCCACCATGGCCAAGTTCGTGGCTGCCTGGACCCTGAAGGCTGCCGCTATGAGTCTTCTAACCGAGGTCGA (SEQ ID NO:66). The 3′ primer is theoligonucleotide TCACTTGAATCGCTGCATCTGCACCCCCAT (SEQ ID NO:67). Influenzavirus from the America Type Tissue Collection (ATCC) is used as a sourcefor the matrix coding region (Perdue et al. Science 279:393-396 (1998)),which is incorporated herein by reference (GenBank accession No.AF036358).

A DNA construct containing a pan DR epitope sequence (SEQ ID NO:52.)fused to the amino-terminus of HBV-S antigen was generated (FIG. 8).This construct is designated PADRE-HBV-s and was generated by annealingtwo overlapping oligonucleotides to add PADRE onto the amino terminus ofhepatitis B surface antigen (Michel et al., Proc. Natl. Acad. Sci. USA81:7708-7712 (1984); Michel et al., Proc. Natl. Acad. Sci. USA92:5307-5311 (1995)), each of which is incorporated herein by reference.One oligonucleotide wasGCTAGCGCCGCCACCATGGCCAAGTTCGTGGCTGCCTGGACCCTGAAGGCTGC CGCTC (SEQ IDNO:68). The second oligonucleotide wasCTCGAGAGCGGCAGCCTTCAGGGTCCAGGCAGCCACGAACTTGGCCATGGTG GCGGCG (SEQ IDNO:69). When annealed, the oligos have NheI and XhoI cohesive ends. Theoligos were heated to 100° C. and slowly cooled to room temperature toanneal. A three part ligation joined PADRE with an XhoI-KpnI fragmentcontaining HBV-s antigen into the NheI plus KpnI sites of the expressionvector.

A DNA construct containing the signal sequence of Ig-α fused to the MHCclass II epitope string shown in FIG. 2 and the transmembrane andcytoplasmic domains of Ig-α was generated (FIG. 9). The mouse Ig-α genehas been described previously (Kashiwamura et al., J. Immunol.145:337-343 (1990)), which is incorporated herein by reference (GenBankaccession No. M31773). This construct, designated Ig-alphaTh, wasconstructed as depicted in FIG. 18. The primer 1F used was theoligonucleotide designated Ig alpha-1F, GCG GCT AGC GCC GCC ACC ATG CCAGGG GGT CTA (SEQ ID NO:70). The primer 1R used was the oligonucleotidedesignated Igalpha-1R, GCA CAG CCT ATG TGA TCG CCT GGC ATC CGG (SEQ IDNO:71). The primer 2F used was the oligonucleotide designatedIgalpha-2F, CTG AAG GCT GCC GCT GGG ATC ATC TTG CTG (SEQ ID NO:72). Theprimer 2R used was the oligonucleotide designated Igalpha-2R, GCG GGTACC TCA TGG CTT TTC CAG CTG (SEQ ID NO:73).

A DNA construct containing the signal sequence of Ig-β fused to the MHCclass II string shown in FIG. 2 and the transmembrane and cytoplasmicdomains of Igβ was generated (FIG. 10). The Ig-β sequence is the B29gene of mouse and has been described previously (Hermanson et al., Proc.Natl. Acad. Sci. USA 85:6890-6894 (1988)), which is incorporated hereinby reference (GenBank accession No. J03857). This construct, designatedIg-betaTh, was constructed as depicted in FIG. 18. The primer 1F usedwas the oligonucleotide designated B29-1F (33mer) GCG GCT AGC GCC GCCACC ATG GCC ACA CTG GTG (SEQ ID NO:74). The primer 1R used was theoligonucleotide designated B29-1R (30mer) CAC AGC CTG GCT GAT CGG CTCACC TGA GAA (SEQ ID NO:75). The primer 2F used was the oligonucleotidedesignated B292F (30mer) CTG AAG GCT GCC GCT ATT ATC TTG ATC CAG (SEQ IDNO:76). The primer 2R used was the oligonucleotide designated B29-2R(27mer), GCC GGT ACC TCA TTC CTG GCC TGG ATG (SEQ ID NO:77).

A DNA construct containing the signal sequence of the kappaimmunoglobulin signal sequence fused to the MHC class II epitope stringshown in FIG. 2 was constructed (FIG. 11). This construct is designatedSigTh and was generated by using the kappaLAMP-Th construct (shown inFIG. 4) and amplifying with the primer pair KappaSig-F (SEQ ID NO:54)plus Help-epR (SEQ ID NO:47) to create SigTh. SigTh contains the kappaimmunoglobulin signal sequence fused to the T helper epitope string andterminated with a translational stop codon.

Constructs encoding human sequences corresponding to the above describedconstructs having mouse sequences are prepared by substituting humansequences for the mouse sequences. Briefly, for the IiPADRE construct,corresponding to FIG. 1, amino acid residues 1-80 from the human Ii geneHLA-DR sequence (FIG. 12) (GenBank accession No. X00497 M14765) issubstituted for the mouse Ii sequences, which is fused to PADRE,followed by human invariant chain HLA-DR amino acid residues 114-223.For the I80T construct, corresponding to FIG. 2, amino acid residues1-80 from the human sequence of Ii is followed by a MHC class II epitopestring. For the IiThfull construct, corresponding to FIG. 3, amino acidresidues 1-80 from the human sequence of Ii, which is fused to a MHCclass II epitope string, is followed by human invariant chain amino acidresidues 114-223.

For the LAMP-Th construct, similar to FIG. 4, the signal sequenceencoded by amino acid residues 11∝19 (nucleotides 11-67) of humanLAMP-1(FIG. 13) (GenBank accession No. J04182), which is fused to theMHC class II epitope string, is followed by the transmembrane(nucleotides 1163-1213) and cytoplasmic tail (nucleotides 1214-1258)region encoded by amino acid residues 380-416 of human LAMP-1.

For the HLA-DM-Th construct, corresponding to FIG. 5, the signalsequence encoded by amino acid residues 1-17 (nucleotides 1-51) of humanHLA-DMB (FIG. 14) (GenBank accession No. U15085), which is fused to theMHC class II epitope string, is followed by the transmembrane(nucleotides 646-720) and cytoplasmic tail (nucleotides 721-792) regionencoded by amino acid residues 216-263 of human HLA-DMB.

For the HLA-DO-Th construct, corresponding to FIG. 6, the signalsequence encoded by amino acid residues 1-21 (nucleotides 1-63) of humanHLA-DO (FIG. 15) (GenBank accession No. L29472 J02736 N00052), which isfused to the MHC class II epitope string, is followed by thetransmembrane (nucleotides 685-735) and cytoplasmic tail (nucleotides736-819) region encoded by amino acid residues 223-273 of human HLA-DO.

For the Ig-alphaTh construct, corresponding to FIG. 9, the signalsequence encoded by amino acid residues 1-29 (nucleotides 1-87) of humanIg-α MB-1 (FIG. 16) (GenBank accession No. U05259), which is fused tothe MHC class II epitope string, is followed by the transmembrane(nucleotides 424-498) and cytoplasmic tail (nucleotides 499-678) regionencoded by amino acid residues 142-226 of human Ig-α MB-1.

For the Ig-betaTh construct, corresponding to FIG. 10, the signalsequence encoded by amino acid residues 1-28 (nucleotides 17-100) ofhuman Ig-β 29 (FIG. 17) (GenBank accession No. M80461), which is fusedto the MHC class II epitope string, is followed by the transmembrane(nucleotides 500-547) and cytoplasmic tail (nucleotides 548-703) regionencoded by amino acid residues 156-229 of human Ig-β.

The SigTh construct shown in FIG. 11 can be used in mouse and human.Alternatively, a signal sequence derived from an appropriate human genecontaining a signal sequence can be substituted for the mouse kappaimmunoglobulin sequence in the Sig Th construct.

The PADRE-Influenza matrix construct shown in FIG. 7 and the PADRE-HBVsconstruct shown in FIG. 8 can be used in mouse and human.

Some of the DNA constructs described above were cloned into the vectorpEP2 (FIG. 19; SEQ ID NO:35). The pEP2 vector was constructed to containdual CMV promoters. The pEP2 vector used the backbone ofpcDNA3.1(−)Myc-His A from Invitrogen and pIRES1hyg from Clontech.Changes were made to both vectors before the CMV transcription unit frompIRES 1 hyg was moved into the modified pcDNA vector.

The pcDNA3.1(−)Myc-His A vector (http://www.invitrogen.com) wasmodified. Briefly, the PvuII fragment (nucleotides 1342-3508) wasdeleted. A BspHI fragment that contains the Ampicillin resistance gene(nucleotides 4404-5412) was cut out. The Ampicillin resistance gene wasreplaced with the kanamycin resistance gene from pUC4K (GenBankAccession #X06404). pUC4K was amplified with the primer set:TCTGATGTTACATTGCACAAG (SEQ ID NO:78) (nucleotides 1621-1601) andGCGCACTCATGATGCTCTGCCAGTGTTACAACC (SEQ ID NO:79) (nucleotides 682-702plus the addition of a BspHI restriction site on the 5′ end). The PCRproduct was digested with BspHI and ligated into the vector digestedwith BspHI. The region between the PmeI site at nucleotide 905 and theEcoRV site at nucleotide 947 was deleted. The vector was then digestedwith PmeI (cuts at nucleotide 1076) and ApaI (cuts at nucleotide 1004),Klenow filled in at the cohesive ends and ligated. The KpnI site atnucleotide 994 was deleted by digesting with KpnI and filling in theends with Klenow DNA polymerase, and ligating. The intron A sequencefrom CMV (GenBank accession M21295, nucleotides 635-1461) was added byamplifying CMV DNA with the primer set: GCGTCTAGAGTAAGTACCGCCTATAGACTC(SEQ ID NO:80) (nucleotides 635-655 plus an XbaI site on the 5′ end) andCCGGCTAGCCTGCAGAAAAGACCCATGGAA (SEQ ID NO:81) (nucleotides 1461-1441plus an NheI site on the 3′ end). The PCR product was digested with XbaIand NheI and ligated into the NheI site of the vector (nucleotide 895 ofthe original pcDNA vector) so that the NheI site was on the 3′ end ofthe intron.

To modify the pIRES1hyg vector (GenBank Accession U89672, Clontech), theKpnI site (nucleotide 911) was deleted by cutting and filling in withKlenow. The plasmid was cut with NotI (nucleotide 1254) and XbaI(nucleotide 3196) and a polylinker oligo was inserted into the site. Thepolylinker was formed by annealing the following two oligos:

GGCCGCAAGGAAAAAATCTAGAGTCGGCCATAGACTAATGCCGGTACCG (SEQ ID NO:82) and

CTAGCGGTACCGGCATTAGTCTATGGCCCGACTCTAGATTTTTTCCTTGC (SEQ ID NO:83). Theresulting plasmid was cut with HincII and the fragment between HincIIsites 234 and 3538 was isolated and ligated into the modified pcDNAvector. This fragment contains a CMV promoter, intron, polylinker, andpolyadenylation signal.

The pIREShyg piece and the pcDNA piece were combined to form pEP2. Themodified pcDNA3.1 (−)Myc-His A vector was partially digested with PvuIIto isolate a linear fragment with the cut downstream of the pcDNApolyadenylation signal (the other PvuII site is the CMV intron). TheHincII fragment from the modified pIRES1hyg vector was ligated into thePvuII cut vector. The polyadenylation signal from the pcDNA derivedtranscription unit was deleted by digesting with EcoRI (pcDNA nucleotide955) and Xhol (pIRES1hyg nucleotide 3472) and replaced with a syntheticpolyadenylation sequence. The synthetic polyadenylation signal wasdescribed in Levitt et al., Genes and Development 3:1019-1025 (1989)).

Two oligos were annealed to produce a fragment that contained apolylinker and polyadenylation signal with EcoRI and XhoI cohesive ends.The oligos were:

AATTCGGATATCCAAGCTTGATGAATAAAAGATCAGAGCTCTAGTGATCTGTGTGTTGGTTTTTTTGTGTGC (SEQ ID NO:84) and

TCGAGCACACAAAAAACCAACACACAGATCACTAGAGCTCTGATCTTTTTATTCATCAAGCTTGGATATCCG (SEQ ID NO:85).

The resulting vector is named pEP2 and contains two separatetranscription units. Both transcription units use the same CMV promoterbut each contains different intron, polylinker, and polyadenylationsequences.

The pEP2 vector contains two transcription units. The firsttranscription unit contains the CMV promoter initially from pcDNA(nucleotides 210-862 in FIG. 19), CMV intron A sequence (nucleotides900-1728 in FIG. 19), polylinker cloning site (nucleotides 1740-1760 inFIG. 19) and synthetic polyadenylation signal (nucleotides 1764-1769 inFIG. 19). The second transcription unit, which was initially derivedfrom pIRES I hyg, contains the CMV promoter (nucleotides 3165-2493 inFIG. 19), intron sequence (nucleotides 2464-2173 in FIG. 19), polylinkerclone site (nucleotides 2126-2095 in FIG. 19) and bovine growth hormonepolyadenylation signal (nucleotides 1979-1974 in FIG. 19). The kanamycinresistance gene is encoded in nucleotides 4965-4061 (FIG. 19).

The DNA constructs described above were digested with NheI and KpnI andcloned into the XbaI and KpnI sites of pEP2 (the second transcriptionunit).

Additional vectors were also constructed. To test for the effect ofco-expression of MHC class I epitopes with MHC class II epitopes, aninsert was generated, designated AOS, that contains nine MHC class Iepitopes. The AOS insert was initially constructed in the vector pMIN.0(FIG. 20; SEQ ID NO:36). Briefly, the AOS insert contains nine MHC classI epitopes, six restricted by HLA-A2 and three restricted by HLA-A11,and the universal MHC class II epitope PADRE. The vector pMIN.0 containsepitopes from HBV, HIV and a mouse ovalbumin epitope. The MHC class Iepitopes appear in pMIN.0 in the following order:

consensus mouse Ig Kappa signal sequence (pMIN.0 amino acid residues1-20, nucleotides 16-81) MQVQIQSLFLLLLWVPGSRG (SEQ ID NO:86) encoded bynucleotides ATG CAG GTG CAG ATC CAG AGC CTG TTT CTG CTC CTC CTG TGG GTGCCC GGG TCC AGA GGA (SEQ ID NO:87);

HBV pol 149-159 (A11 restricted)

(pMIN.0 amino acid residues 21-31, nucleotides 82-114) HTLWKAGILYK (SEQID NO:88) encoded by nucleotides CAC ACC CTG TGG AAG GCC GGAATC CTG TATAAG (SEQ ID NO:89);

PADRE-universal MHC class II epitope (pMIN.0 amino acid residues 32-45,nucleotides 115-153) AKFVAAWTLKAAA (SEQ ID NO:52) encoded by nucleotidesGCC AAG TTC GTG GCT GCC TGG ACC CTG AAG GCT GCC GCT (SEQ ID NO:90);

HBV core 18-27 (A2 restricted) (pMIN.0 amino acid residues 46-55,nucleotides 154-183) FLPSDFFPSV (SEQ ID NO:91) encoded by nucleotidesTTC CTG CCT AGC GAT TTC TTT CCT AGC GTG (SEQ ID NO:92);

HIV env 120-128 (A2 restricted) (pMIN.0 amino acid residues 56-64,nucleotides 184-210) KLTPLCVTL (SEQ ID NO:93) encoded by nucleotides AAGCTG ACC CCA CTG TGC GTG ACC CTG (SEQ ID NO:94);

HBV pol 551-559 (A2 restricted) (pMIN.0 amino acid residues 65-73,nucleotides 211-237) YMDDVVLGA (SEQ ID NO:95) encoded by nucleotides TATATG GAT GAC GTG GTG CTG GGA GCC (SEQ ID NO:96);

mouse ovalbumin 257-264 (K^(b) restricted) (pMIN.0 amino acid residues74-81, nucleotides 238-261) SIINFEKL (SEQ ID NO:97) encoded bynucleotides AGC ATC ATC AAC TTC GAG AAG CTG (SEQ ID NO:98);

HBV pol 455-463 (A2 restricted) (pMIN.0 amino acid residues 82-90,nucleotides 262-288) GLSRYVARL (SEQ ID NO:99) encoded by nucleotides GGACTG TCC AGA TAC GTG GCT AGG CTG (SEQ ID NO:100);

HIV pol 476-84 (A2 restricted) (pMIN.0 amino acid residues 91-99,nucleotides 289-315) ILKEPVHGV (SEQ ID NO:101) encoded by nucleotidesATC CTG AAG GAG CCT GTG CAC GGC GTG (SEQ ID NO:102);

HBV core 141-151 (All restricted)

(pMIN.0 amino acid residues 100-110, nucleotides 316-348) STLPETTVVRR(SEQ ID NO:103) encoded by nucleotides TCC ACC CTG CCA GAG ACC ACC GTGGTG AGG AGA (SEQ ID NO:104);

HIV env 49-58 (All restricted) (pMIN.0 amino acid residues 111-120,nucleotides 349-378) TVYYGVPVWK (SEQ ID NO:105) encoded by nucleotidesACC GTG TAC TAT GGA GTG CCT GTG TGG AAG (SEQ ID NO:106); and

HBV env 335-343 (A2 restricted) (pMIN.0 amino acid residues 121-129,nucleotides 378-405) WLSLLVPFV (SEQ ID NO:107) encoded by nucleotidesTGG CTG AGC CTG CTG GTG CCC TTT GTG (SEQ ID NO:108).

The pMIN.0 vector contains a KpnI restriction site (pMIN.0 nucleotides406-411) and a NheI restriction site (pMIN.0 nucleotides 1-6). ThepMIN.0 vector contains a consensus Kozak sequence (nucleotides 7-18)(GCCGCCACCATG; SEQ ID NO:109) and murine Kappa Ig-light chain signalsequence followed by a string of 10 MHC class I epitopes and oneuniversal MHC class II epitope. The pMIN.0 sequence encodes an openreading frame fused to the Myc and His antibody epitope tag coded for bythe pcDNA 3.1 Myc-His vector. The pMIN.0 vector was constructed witheight oligonucleotides:

Min1 oligo

GAGGAGCAGAAACAGGCTCTGGATCTGCACCTGCATTCCCATGGTGGCGGCGC TAGCAAGCTTCTTGCGC(SEQ ID NO:110);

Min2 oligo

CCTGTTTCTGCTCCTCCTGTGGGTGCCCGGGTCCAGAGGACACACCCTGTGGA AGGCCGGAATCCTGTATA(SEQ ID NO:11);

Min3 oligo

TCGCTAGGCAGGAAAGCGGCAGCCTTCAGGGTCCAGGCAGCCACGAACTTGG CCTTATACAGGATTCCGG(SEQ ID NO:112);

Min4 oligo

CTTTCCTGCCTAGCGATTTCTTTCCTAGCGTGAAGCTGACCCCACTGTGCGTGA CCCTGTATATGGATGAC(SEQ ID NO:113);

Min5 oligo

CGTACCTGGACAGTCCCAGCTTCTCGAAGTTGATGATGCTGGCT CCCAGCACCACGTCATCCATATACAG(SEQ ID NO:114);

Min6 oligo

GGACTGTCCAGATACGTGGCTAGGCTGATCCTGAAGGAGCCTGTGCACGGCGT GTCCACCCTGCCAGAGAC(SEQ ID NO:115);

Min7 oligo

GCTCAGCCACTTCCACACAGGCACTCCATAGTACACGGTCCTCCTCACCACGG TGGTCTCTGGCAGGGTG(SEQ ID NO:116);

Min8 oligo

GTGGAAGTGGCTGAGCCTGCTGGTGCCCTTTGTGGGTACCTGATCTAGAGC (SEQ ID NO:117).

Additional primers were flanking primer 5′, GCG CAA GAA GCT TGC TAG CG(SEQ ID NO:118) and flanking primer 3′, GCT CTA GAT CAG GTA CCC CAC (SEQID NO:119).

The original pMIN.0 minigene construction was carried out using eightoverlapping oligos averaging approximately 70 nucleotides in length,which were synthesized and HPLC purified by Operon Technologies Inc.Each oligo overlapped its neighbor by 15 nucleotides, and the finalmulti-epitope minigene was assembled by extending the overlapping oligosin three sets of reactions using PCR (Ho et al., Gene 77:51-59 (1989).

For the first PCR reaction, 5 μg of each of two oligos were annealed andextended: 1+2, 3+4, 5+6, and 7+8 were combined in 100 μl reactionscontaining 0.25 mM each dNTP and 2.5 units of Pfu polymerase in Pfupolymerase buffer containing 10 mM KCl, 10 mM (NH₄)₂SO₄, 20 mMTris-chloride, pH 8.75, 2 mM MgSO₄, 0.1% TRITON X-100 and 100 mg/ml BSA.A Perkin/Elmer 9600 PCR machine was used and the annealing temperatureused was 5° C. below the lowest calculated T_(m) of each primer pair.The full length dimer products were gel-purified, and two reactionscontaining the product of 1-2 and 3-4, and the product of 5-6 and 7-8were mixed, annealed and extended for 10 cycles. Half of the tworeactions were then mixed, and 5 cycles of annealing and extensioncarried out before flanking primers were added to amplify the fulllength product for 25 additional cycles. The full length product was gelpurified and cloned into pCR-blunt (Invitrogen) and individual cloneswere screened by sequencing. The Min insert was isolated as an NheI-KpnIfragment and cloned into the same sites of pcDNA3.1(−)/Myc-His A(Invitrogen) for expression. The Min protein contains the Myc and Hisantibody epitope tags at its carboxyl-terminal end.

For all the PCR reactions described, a total of 30 cycles were performedusing Pfu polymerase and the following conditions: 95° C. for 15seconds, annealing temperature for 30 seconds, 72° C. for one minute.The annealing temperature used was 5° C. below the lowest calculated Tmof each primer pair.

Three changes to pMIN.0 were made to produce pMIN.1 (FIG. 21; SEQ IDNO:37, also referred to as pMIN-AOS). The mouse ova epitope was removed,the position 9 alanine anchor residue (#547) of HBV pol 551-560 wasconverted to a valine which increased the in vitro binding affinity40-fold, and a translational stop codon was introduced at the end of themulti-epitope coding sequence. The changes were made by amplifying twooverlapping fragments and combining them to yield the full lengthproduct.

The first reaction used the 5′ pcDNA vector primer T7 and the primerMin-ovaR (nucleotides 247-218) TGGACAGTCCCACTCCCAGCACCACGTCAT (SEQ IDNO:120). The 3′ half was amplified with the primers: Min-ovaF(nucleotides 228-257) GCTGGGAGTGGGACTGTCCAGGTACGTGGC (SEQ ID NO:121) andMin-StopR (nucleotides 390-361) GGTACCTCACACAAAGGGCACCAGCAGGC (SEQ IDNO:122)

The two fragments were gel purified, mixed, denatured, annealed, andfilled in with five cycles of PCR. The full length fragment wasamplified with the flanking primers T7 and Min-Stop for 25 more cycles.The product was gel purified, digested with NheI and KpnI and clonedinto pcDNA3.1 for sequencing and expression. The insert from pMin.1 wasisolated as an NheI-KpnI fragment and cloned into pEP2 to make pEP2-AOS.

Example II Assay for T Helper Cell Activation

This example shows methods for assaying T helper cell activity. Onemethod for assaying T helper cell activity uses spleen cells of animmunized organism. Briefly, a spleen cell pellet is suspended with 2-3ml of red blood cell lysis buffer containing 8.3 g/liter ammoniumchloride in 0.001 M Tris-HCl, pH 7.5. The cells are incubated in lysisbuffer for 3-5 min at room temperature with occasional vortexing. Anexcess volume of 50 ml of R10 medium is added to the cells, and thecells are pelleted. The cells are resuspended and pelleted one or twomore times in R2 medium or R10 medium.

The cell pellet is suspended in R10 medium and counted. If the cellsuspension is aggregated, the aggregates are removed by filtration or byallowing the aggregates to settle by gravity. The cell concentration isbrought to 10⁷/ml, and 100 μl of spleen cells are added to 96 well flatbottom plates.

Dilutions of the appropriate peptide, such as pan DR epitope (SEQ IDNO:145), are prepared in R10 medium at 100, 10, 1, 0.1 and 0.01 μg/ml,and 100 μl of peptide are added to duplicate or triplicate wells ofspleen cells. The final peptide concentration is 50, 5, 0.5, 0.05 and0.005 μg/ml. Control wells receive 100 μl R10 medium.

The plates are incubated for 3 days at 37° C. After 3 days, 20 μl of 50μCi/ml ³H-thymidine is added per well. Cells are incubated for 18-24hours and then harvested onto glass fiber filters. The incorporation of³H-thymidine into DNA of proliferating cells is measured in a betacounter.

A second assay for T helper cell activity uses peripheral bloodmononuclear cells (PBMC) that are stimulated in vitro as described inAlexander et al., supra and Sette (WO 95/07,707), as adapted from Mancaet al., J. Immunol. 146:1964-1971 (1991), which is incorporated hereinby reference. Briefly, PBMC are collected from healthy donors andpurified over Ficoll-Plaque (Pharmacia Biotech; Piscataway, N.J.). PBMCare plated in a 24 well tissue culture plate at 4×10⁶ cells/ml. Peptidesare added at a final concentration of 10 μg/ml. Cultures are incubatedat 37° C. in 5% CO₂.

On day 4, recombinant interleukin-2 (IL-2) is added at a finalconcentration of 10 ng/ml. Cultures are fed every 3 days by aspirating 1ml of medium and replacing with fresh medium containing IL-2. Twoadditional stimulations of the T cells with antigen are performed onapproximately days 14 and 28. The T cells (3×10 ⁵/well) are stimulatedwith peptide (10 μg/ml) using autologous PBMC cells (2×10⁶ irradiatedcells/well) (irradiated with 7500 rads) as antigen-presenting cells in atotal of three wells of a 24 well tissue culture plate. In addition, onday 14 and 28, T cell proliferative responses are determined under thefollowing conditions: 2×10⁴ T cells/well; 1×10⁵ irradiated PBMC/well asantigen-presenting cells; peptide concentration varying between 0.01 and10 μg/ml final concentration. The proliferation of the T cells ismeasured 3 days later by the addition of ³H-thymidine (1 μCi/well) 18 hrprior to harvesting the cells. Cells are harvested onto glass filtersand ³H-thymidine incorporation is measured in a beta plate counter.These results demonstrate methods for assaying T helper cell activity bymeasuring ³H-thymidine incorporation.

Example III Assay for Cytotoxic T Lymphocyte Response

This example shows a method for assaying cytotoxic T lymphocyte (CTL)activity. A CTL response is measured essentially as described previously(Vitiello et al., Eur. J. Immunol. 27:671-678 (1997), which isincorporated herein by reference). Briefly, after approximately 10-35days following DNA immunization, splenocytes from an animal are isolatedand co-cultured at 37° C. with syngeneic, irradiated (3000 rad)peptide-coated LPS blasts (1×10⁶ to 1.5×10⁶ cells/ml) in 10 ml R10 inT25 flasks. LPS blasts are obtained by activating splenocytes (1×10⁶ to1.5×10⁶ cells/ml) with 25 μg/ml lipopolysaccharides (LPS) (Sigma cat.no. L-2387; St. Louis, Mo.) and 7 μg/ml dextran sulfate (PharmaciaBiotech) in 30 ml R10 medium in T75 flasks for 3 days at 37° C. Thelymphoblasts are then resuspended at a concentration of 2.5×10⁷ to3.0×10⁷/ml, irradiated (3000 rad), and coated with the appropriatepeptides (100 μg/ml) for 1 h at 37° C. Cells are washed once,resuspended in R10 medium at the desired concentration and added to theresponder cell preparation. Cultures are assayed for cytolytic activityon day 7 in a ⁵¹Cr-release assay.

For the ⁵¹Cr-release assay, target cells are labeled for 90 min at 37°C. with 150 μsodium ⁵¹chromate (⁵¹Cr) (New England Nuclear; WilmingtonDel.), washed three times and resuspended at the appropriateconcentration in R10 medium. For the assay, 10⁴ target cells areincubated in the presence of different concentrations of effector cellsin a final volume of 200 μl in U-bottom 96 well plates in the presenceor absence of 10 μg/ml peptide. Supernatants are removed after 6 h at37° C., and the percent specific lysis is determined by the formula:percent specific lysis=100×(experimental release−spontaneousrelease)/(maximum release−spontaneous release). To facilitate comparisonof responses from different experiments, the percent release data istransformed to lytic units 30 per 10⁶ cells (LU30/10⁶), with 1 LU30defined as the number of effector cells required to induce 30% lysis of10⁴ target cells in a 6 h assay. LU values represent the LU30/10⁶obtained in the presence of peptide minus LU30/10⁶ in the absence ofpeptide. These results demonstrate methods for assaying CTL activity bymeasuring ⁵¹Cr release from cells.

Example IV T Cell Proliferation in Mice Immunized with ExpressionVectors Encoding MHC Class II Epitopes and MHC Class II TargetingSequences

This example demonstrates that expression vectors encoding MHC class IIepitopes and MHC class II targeting sequences are effective atactivating T cells.

The constructs used in the T cell proliferation assay are described inExample I and were cloned into the vector pEP2, a CMV driven expressionvector. The peptides used for T cell in vitro stimulation are: Ova323-339, ISQAVHAAHAEINEAGR (SEQ ID NO:48); HBVcore128, TPPAYRPPNAPILF(SEQ ID NO:124); HBVenv182, FFLLTRILTIPQSLD (SEQ ID NO:-50); and PADRE,AKFVAAWTLKAAA (SEQ ID NO:52).

T cell proliferation was assayed essentially as described in Example II.Briefly, 12 to 16 week old B6D2 F1 mice (2 mice per construct) wereinjected with 100 μg of the indicated expression vector (50 μg per leg)in the anterior tibialis muscle. After eleven days, spleens werecollected from the mice and separated into a single cell suspension byDounce homogenization. The splenocytes were counted and one millionsplenocytes were plated per well in a 96-well plate. Each sample wasdone in triplicate. Ten μg/ml of the corresponding peptide encoded bythe respective expression vectors was added to each well. One wellcontained splenocytes without peptide added for a negative control.Cells were cultured at 37° C., 5% CO₂ for three days.

After three days, one μCi of ³H-thymidine was added to each well. After18 hours at 37° C., the cells were harvested onto glass filters and ³Hincorporation was measured on an LKB β plate counter. The results of theT cell proliferation assay are shown in Table 9. Antigenspecific T cellproliferation is presented as the stimulation index (SI); this isdefined as the ratio of the average ³H-thymidine incorporation in thepresence of antigen divided by the ³H-thymidine incorporation in theabsence of antigen.

The immunogen “PADRE+IFA” is a positive control where the PADRE peptidein incomplete Freund's adjuvant was injected into the mice and comparedto the response seen by injecting the MHC class II epitope constructscontaining a PADRE sequence. As shown in Table 9, most of the expressionvectors tested were effective at activating T cell proliferation inresponse to the addition of PADRE peptide. The activity of several ofthe expression vectors was comparable to that seen with immunizationwith the PADRE peptide in incomplete Freund's adjuvant. The expressionvectors containing both MHC class I and MHC class II epitopes, pEP2-AOSand pcDNA-AOS, were also effective at activating T cell proliferation inresponse to the addition of PADRE peptide.

These results show that expression vectors encoding MHC class IIepitopes fused to a MHC class II targeting sequence is effective atactivating T cell proliferation and are useful for stimulating an immuneresponse.

Example V In vivo Assay Using Transgenic Mice

A. Materials and Methods

Peptides were synthesized according to standard F-moc solid phasesynthesis methods which have been previously described (Ruppert et al.,Cell 74:929 (1993); Sette et al., Mol. Immunol. 31:813 (1994)). Peptidepurity was determined by analytical reverse-phase HPLC and purity wasroutinely>95%. Synthesis and purification of the Theradigm-HBVlipopeptide vaccine is described in (Vitiello et al., J. Clin. Invest.95:341 (1995)).

Mice

HLA-A2.1 transgenic mice used in this study were the F1 generationderived by crossing transgenic mice expressing a chimeric geneconsisting of the α1, α2 domains of HLA-A2.1 and α3 domain of H-2K^(b)with SJL/J mice (Jackson Laboratory, Bar Harbor, Me.). This strain willbe referred to hereafter as HLA-A2.1/K^(b)-H-2^(bxs). The parentalHLA-A2.1/K^(b) transgenic strain was generated on a C57BL/6 backgroundusing the transgene and methods described in (Vitiello et al., J. Exp.Med. 173:1007 (1991)). HLA-A11/K^(b) transgenic mice used in the currentstudy were identical to those described in (Alexander et al., J.Immunol. 159:4753 (1997)).

Cell lines. MHC Purification, and Peptide Binding Assay

Target cells for peptide-specific cytotoxicity assays were Jurkat cellstransfected with the HLA-A2.1/K^(b) chimeric gene (Vitiello et al., J.Exp. Med. 173:1007 (1991)) and .221 tumor cells transfected withHLA-A11/K^(b) (Alexander et al., J. Immunol. 159:4753 (1997)).

To measure presentation of endogenously processed epitopes,Jurkat-A2.1/K^(b) cells were transfected with the pMin.1 or pMin.2-GFPminigenes then tested in a cytotoxicity assay against epitope-specificCTL lines. For transfection, Jurkat-A2.1/K^(b) cells were resuspended at10⁷ cells/ml and 30 μg of DNA was added to 600 μl of cell suspension.After electroporating cells in a 0.4 cm cuvette at 0.25 kV, 960 μFd,cells were incubated on ice for 10 min then cultured for 2 d in RPMIculture medium. Cells were then cultured in medium containing 200 U/mlhygromycin B (Calbiochem, San Diego Calif.) to select for stabletransfectants. FACS was used to enrich the fraction of green fluorescentprotein (GFP)-expressing cells from 15% to 60% (data not shown).

Methods for measuring the quantitative binding of peptides to purifiedHLA-A2.1 and -A11 molecules is described in Ruppert et al., Cell 74:929(1993); Sette et al., Mol. Immunol. 31:813 (1994); Alexander et al., J.Immunol. 159:4753 (1997).

All tumor cell lines and splenic CTLs from primed mice were grown inculture medium (CM) that consisted of RPMI 1640 medium with Hepes (LifeTechnologies, Grand Island, N.Y.) supplemented with 10% FBS, 4 mML-glutamine, 5×10⁻⁵ M 2-ME, 0.5 mM sodium pyruvate, 100 μg/mlstreptomycin, and 100 U/ml penicillin.

Construction of Minigene Multi-epitope DNA Plasmids

pMIN.0 and pMIN.1 (i.e., pMIN-AOS) were constructed as described aboveand in U.S. Ser. No. 60/085,751.

pMin.1-No PADRE and pMin.1-Anchor. pMin.1 was amplified using twooverlapping fragments which was then combined to yield the full lengthproduct. The first reaction used the 5′ pcDNA vector primer T7 andeither primer ATCGCTAGGCAGGAACTTATACAGGATTCC (SEQ ID NO:126) for pMin.1-No PADRE or TGGACAGTCCGGCTCCCAGCACCACGT (SEQ ID NO:127) for pMin.1-Anchor. The 3′ half was amplified with the primers TTCCTGCCTAGCGATTTC(SEQ ID NO:128) (No PADRE) or GCTGGGAGCCGGACTGTCCAGGTACGT (SEQ IDNO:129) (Anchor) and Min-StopR. The two fragments generated fromamplifying the 5′ and 3′ ends were gel purified, mixed, denatured,annealed, and filled in with five cycles of PCR. The full lengthfragment was further amplified with the flanking primers T7 andMin-StopR for 25 more cycles.

pMin.1-No Sig. The Ig signal sequence was deleted from pMin.1 by PCRamplification with primer GCTAGCGCCGCCACCATGCACACCCTGTGGAAGGC CGGAATC(SEQ ID NO:130) and pcDNA rev (Invitrogen) primers. The product wascloned into pCR-blunt and sequenced.

pMin.1-Switch. Three overlapping fragments were amplified from pMin.1,combined, and extended. The 5′ fragment was amplified with the vectorprimer T7 and primer GGGCACCAGCAGGCTCAGCCACACTCCCAGCACCACGTC (SEQ IDNO:131). The second overlapping fragment was amplified with primersAGCCTGCTGGTGCCCTTTGTGATCCTGAAGGAGCCTGTGC (SEQ ID NO:132) andAGCCACGTACCTGGACAGTCCCTTCCACACAGGCACTCCAT (SEQ ID NO:133). PrimerTGTCCAGGTACGTGGCTAGGCTGTGAGGTACC (SEQ ID NO:134) and the vector primerpcDNA rev (Invitrogen) were used to amplify the third (3′) fragment.Fragments 1, 2, and 3 were amplified and gel purified. Fragments 2 and 3were mixed, annealed, amplified, and gel purified. Fragment 1 wascombined with the product of 2 and 3, and extended, gel purified andcloned into pcDNA3.1 for expression.

pMin.2-GFP. The signal sequence was deleted from pMin.0 by PCRamplification with Min.0-No Sig-5′GCTAGCGCCGCCACCATGCACACCCTGTGGAAGGCCGGAATC (SEQ ID NO:135) plus pcDNArev (Invitrogen) primers. The product was cloned into pCR-blunt andsequenced. The insert containing the open reading frame of the signalsequence-deleted multi-epitope construct was cut out with NheI plusHindIII and ligated into the same sites of pEGFPN1 (Clontech). Thisconstruct fuses the coding region of the signal-deleted pMin.0 constructto the N-terminus of green fluorescent protein (GFP).

Immunization of Mice

For DNA immunization, mice were pretreated by injecting 50 μl of 10 μMcardiotoxin (Sigma Chem. Co., #C9759) bilaterally into the tibialisanterior muscle. Four or five days later, 100 μg of DNA diluted in PBSwere injected in the same muscle.

Theradigm-HBV lipopeptide (10 mg/ml in DMSO) that was stored at−20° C.,was thawed for 10 min at 45° C. before being diluted 1:10 (v/v) withroom temperature PBS. Immediately upon addition of PBS, the lipopeptidesuspension was vortexed vigorously and 100 μl was injected s.c. at thetail base (100 μg/mouse).

Immunogenicity of individual CTL epitopes was tested by mixing each CTLepitope (50 μg/mouse) with the HBV core 128-140 peptide (TPPAYRPPNAPIL(SEQ ID NO:49), 140 μg/mouse) which served to induce I-A^(b)-restrictedTh cells. The peptide cocktail was then emuslifed in incomplete Freund'sadjuvant (Sigma Chem. Co.) and 100 μl of peptide emulsion was injecteds.c. at the tail base.

In vitro CTL Cultures and Cytotoxicity Assays

Eleven to 14 days after immunization, animals were sacrificed and asingle cell suspension of splenocytes prepared. Splenocytes fromcDNA-primed animals were stimulated in vitro with each of the peptideepitopes represented in the minigene. Splenocytes (2.5-3.0×10⁷/flask)were cultured in upright 25 cm² flasks in the presence of 10 μg/mlpeptide and 100 irradiated spleen cells that had been activated for 3days with LPS (25 μg/ml) and dextran sulfate (7 μg/ml). Triplicatecultures were stimulated with each epitope. Five days later, cultureswere fed with fresh CM. After 10 d of in vitro culture, 2-4×10⁶ CTLsfrom each flask were restimulated with 10⁷ LPS/dextran sulfate-activatedsplenocytes treated with 100 μg/ml peptide for 60-75 min at 37° C., thenirradiated 3500 rads. CTLs were restimulated in 6-well plates in 8 ml ofcytokine-free CM. Eighteen hr later, cultures received cytokinescontained in con A-activated splenocyte supernatant (10-15% finalconcentration, v/v) and were fed or expanded on the third day with CMcontaining 10-15% cytokine supernate. Five days after restimulation, CTLactivity of each culture was measured by incubating varying numbers ofCTLs with 10⁴ ⁵¹° C.r-labelled target cells in the presence or absenceof peptide. To decrease nonspecific cytotoxicity from NK cells, YAC-1cells (ATCC) were also added at a YAC-1:⁵¹Cr-labeled target cell ratioof 20:1. CTL activity against the HBV Pol 551 epitope was measured bystimulating DNA-primed splenocytes in vitro with the native A-containingpeptide and testing for cytotoxic activity against the same peptide.

To more readily compare responses, the standard E:T ratio vs %cytotoxicity data curves were converted into LU per 10⁶ effector cellswith one LU defined as the lytic activity required to achieve 30% lysisof target cells at a 100:1 E:T ratio. Specific CTL activity (ΔLU) wascalculated by subtracting the LU value obtained in the absence ofpeptide from the LU value obtained with peptide. A given culture wasscored positive for CTL induction if all of the following criteria weremet: 1) ΔLU>2;2) LU(+peptide)÷LU(−peptide)>3; and 3) a>10% difference in% cytotoxicity tested with and without peptide at the two highest E:Tratios (starting E:T ratios were routinely between 25-50:1).

CTL lines were generated from pMin.1-primed splenocytes through repeatedweekly stimulations of CTLs with peptide-treated LPS/DxS-activatedsplenocytes using the 6-well culture conditions described above with theexception that CTLs were expanded in cytokine-containing CM as necessaryduring the seven day stimulation period.

Cytokine Assay

To measure IFN-γ production in response to minigene-transfected targetcells, 4×10⁴ CTLs were cultured with an equivalent number ofminigene-transfected Jurkat-A2.1/K^(b) cells in 96-well flat bottomplates. After overnight incubation at 37° C., culture supernatant fromeach well was collected and assayed for IFN-γ concentration using asandwich ELISA. Immulon II microtiter wells (Dynatech, Boston, Mass.)were coated overnight at 4° C. with 0.2 μg of anti-mouse IFN-γ captureAb, R4-6A2 (Pharmingen). After washing wells with PBS/0.1% Tween-20 andblocking with 1% BSA, Ab-coated wells were incubated with culturesupernate samples for 2 hr at room temperature. A secondary anti-IFN-γAb, XMG1.2 (Pharmingen), was added to wells and allowed to incubate for2 hr at room temperature. Wells were then developed by incubations withAvidin-DH and finally with biotinylated horseradish peroxidase H(Vectastain ABC kit, Vector Labs, Burlingame, Calif.) and TMB peroxidasesubstrate (Kirkegaard and Perry Labs, Gaithersberg, Md.). The amount ofcytokine present in each sample was calculated using a rIFN-γ standard(Pharmingen).

b. Results

Selection of Epitopes and Minigene Construct Design

In the first series of experiments, the issue was whether a balancedmultispecific CTL response could be induced by simple minigene cDNAconstructs that encode several dominant HLA class I-restricted epitopes.Accordingly, nine CTL epitopes were chosen on the basis of theirrelevance in CTL immunity during HBV and HIV infection in humans, theirsequence conservancy among viral subtypes, and their class I MHC bindingaffinity (Table 10). Of these nine epitopes, six are restricted byHLA-A2.1 and three showed HLA-Al 11-restriction. One epitope, HBV Pol551, was studied in two alternative forms: either the wild type sequenceor an analog (HBV Pol 551-V) engineered for higher binding affinity.

As referenced in Table 10, several independent laboratories havereported that these epitopes are part of the dominant CTL responseduring HBV or HIV infection. All of the epitopes considered showedgreater than 75% conservancy in primary amino acid sequence among thedifferent HBV subtypes and HIV clades. The MHC binding affinity of thepeptides was also considered in selection of the epitopes. Theseexperiment addressed the feasibility of immunizing with epitopespossessing a wide range of affinities and, as shown in Table 10, the sixHBV and three HIV HLA-restricted epitopes covered a spectrum of MHCbinding affinities spanning over two orders of magnitude, with IC₅₀%concentrations ranging from 3 nM to 200 nM.

The immunogenicity of the six A2.1- and three A11-restricted CTLepitopes in transgenic mice was verified by co-immunization with ahelper T cell peptide in an IFA formulation. All of the epitopes inducedsignificant CTL responses in the 5 to 73 ΔLU range (Table 10). Asmentioned above, to improve the MHC binding and immunogenicity of HBVPol 551, the C-terminal A residue of this epitope was substituted with Vresulting in a dramatic 40-fold increase in binding affinity to HLA-A2.1(Table 10). While the parental sequence was weakly or nonimmunogenic inHLA transgenic mice, the HBV Pol 551-V analog induced significant levelsof CTL activity when administered in IFA (Table 10). On the basis ofthese results, the V analog of the HBV Pol 551 epitope was selected forthe initial minigene construct. In all of the experiments reportedherein, CTL responses were measured with target cells coated with thenative HBV Pol 551 epitope, irrespective of whether the V analog ornative epitope was utilized for immunization.

Finally, since previous studies indicated that induction of T cell helpsignificantly improved the magnitude and duration of CTL responses(Vitiello et al., J. Clin. Invest. 95.341 (1995); Livingston et al., J.Immunol. 159:1383 (1997)), the universal Th cell epitope PADRE was alsoincorporated into the minigene. PADRE has been shown previously to havehigh MHC binding affinity to a wide range of mouse and human MHC classII haplotypes (Alexander et al., Immunity 1:751 (1994)). In particular,it has been previously shown that PADRE is highly immunogenic in H-2^(b)mice that are used in the current study (Alexander et al., Immunity1:751 (1994)).

pMin. 1, the prototype cDNA minigene construct encoding nine CTLepitopes and PADRE, was synthesized and subcloned into the pcDNA3.1vector. The position of each of the nine epitopes in the minigene wasoptimized to avoid junctional mouse H-2^(b) and HLA-A2.1 class I MHCepitopes. The mouse Ig K signal sequence was also included at the 5′ endof the construct to facilitate processing of the CTL epitopes in theendoplasmic reticulum (ER) as reported by others (Anderson et al., J.Exp. Med. 174:489 (1991)). To avoid further conformational structure inthe translated polypeptide gene product that may affect processing ofthe CTL epitopes, an ATG stop codon was introduced at the 3′ end of theminigene construct upstream of the coding region for c-myc and poly-hisepitopes in the pcDNA3.1 vector.

Immunopenicity of pMin.1 in Transgenic Mice

To assess the capacity of the pMin.1 minigene construct to induce CTLsin vivo, HLA-A2.1/K^(b)-H-2^(bxs) transgenic mice were immunizedintramuscularly with 100 μg of naked cDNA. As a means of comparing thelevel of CTLs induced by cDNA immunization, a control group of animalswas also immunized with Theradigm-HBV, a palmitolyated lipopeptideconsisting of the HBV Core 18 CTL epitope linked to the tetanus toxin830-843 Th cell epitope.

Splenocytes from immunized animals were stimulated twice with each ofthe peptide epitopes encoded in the minigene, then assayed forpeptide-specific cytotoxic activity in a ⁵¹Cr release assay. Arepresentative panel of CTL responses of pMin.1-primed splenocytes,shown in FIG. 22, clearly indicates that significant levels of CTLinduction were generated by minigene immunization. The majority of thecultures stimulated with the different epitopes exceeded 50% specificlysis of target cells at an E:T ratio of 1:1. The results of fourindependent experiments, compiled in Table 11, indicate that the pMin.1construct is indeed highly immunogenic in HLA-A2.1/K^(b)-H-2^(bxs)transgenic mice, inducing a broad CTL response directed against each ofits six A2.1-restricted epitopes.

To more conveniently compare levels of CTL induction among the differentepitopes, the % cytotoxicity values for each splenocyte culture wasconverted to ΔLU and the mean ΔLU of CTL activity in positive culturesfor each epitope was determined (see Example V, materials and methods,for positive criteria). The data, expressed in this manner in Table 11,confirms the breadth of CTL induction elicited by pMin.1 immunizationsince extremely high CTL responses, ranging between 50 to 700 ΔLU, wereobserved against the six A2.1-restricted epitopes. More significantly,the responses of several hundred ΔLU observed for five of the sixepitopes approached or exceeded that of the Theradigm-HBV lipopeptide, avaccine formulation known for its high CTL-inducing potency (Vitiello etal., J. Clin. Invest. 95:341 (1995); Livingston et al., J. Immunol.159:1383 (1997)). The HBV Env 335 epitope was the only epitope showing alower mean ΔLU response compared to lipopeptide (Table 11, 44 vs 349ΔLU).

Processing of Minigene Epitopes by Transfected Cells

The decreased CTL response observed against HBV Env 335 was somewhatunexpected since this epitope had good A2.1 binding affinity (IC50%, 5nM) and was also immunogenic when administered in IFA. The lowerresponse may be due, at least in part, to the inefficient processing ofthis epitope from the minigene polypeptide by antigen presenting cellsfollowing in vivo cDNA immunization. To address this possibility,Jurkat-A2.1/K^(b) tumor cells were transfected with pMin.1 cDNA and thepresentation of the HBV Env 335 epitope by transfected cells wascompared to more immunogenic A2.1-restricted epitopes using specific CTLlines. Epitope presentation was also studied using tumor cellstransfected with a control cDNA construct, pMin.2-GFP, that encoded asimilar multi-epitope minigene fused with GFP which allows detection ofminigene expression in transfected cells by FACS.

Epitope presentation of the transfected Jurkat cells was analyzed usingspecific CTL lines, with cytotoxicity or IFN-γ production serving as aread-out. It was found that the levels of CTL response correlateddirectly with the in vivo immunogenicity of the epitopes. Highlyimmunogenic epitopes in vivo, such as HBV Core 18, HIV Pol 476, and HBVPol 455, were efficiently presented to CTL lines by pMin.1- orpMin.2-GFP-transfected cells as measured by IFN-γ production (FIG.23A, >100 pg/ml for each epitope) or cytotoxic activity (FIG. 23C,>30%specific lysis). In contrast to these high levels of in vitro activity,the stimulation of the HBV Env 335-specific CTL line against bothpopulations of transfected cells resulted in less than 12 pg/ml IFN-γand 3% specific lysis. Although the HBV Env 335-specific CTL line didnot recognize the naturally processed epitope efficiently, this line didshow an equivalent response to peptide-loaded target cells, as comparedto CTL lines specific for the other epitopes (FIG. 23B, D).Collectively, these results suggest that a processing and/orpresentation defect associated with the HBV Env 335 epitope that maycontribute to its diminished immunogenicity in vivo.

Effect of the Helper T Cell Epitope PADRE on Minigene Immunogenicity

Having obtained a broad and balanced CTL response in transgenic miceimmunized with a minigene cDNA encoding multiple HLA-A2.1-restrictedepitopes, next possible variables were examined that could influence theimmunogenicity of the prototype construct. This type of analysis couldlead to rational and rapid optimization of future constructs. Morespecifically, a cDNA construct based on the pMin.1 prototype wassynthesized in which the PADRE epitope was deleted to examine thecontribution of T cell help in minigene immunogenicity (FIG. 24A).

The results of the immunogenicity analysis indicated that deletion ofthe PADRE Th cell epitope resulted in significant decreases in thefrequency of specific CTL precursors against four of the minigeneepitopes (HBV Core 18, HIV Env 120, HBV Pol 455, and HBV Env 335) asindicated by the 17 to 50% CTL-positive cultures observed against theseepitopes compared to the 90-100% frequency in animals immunized with theprototype pMin.1 construct (FIG. 25). Moreover, for two of the epitopes,HBV Core 18 and HIV Env 120, the magnitude of response in positivecultures induced by pMin.1-No PADRE was 20- to 30-fold less than that ofthe pMin. 1 construct (FIG. 25A).

Effect of Modulation of MHC Binding Affinity on Epitope Immunogenicity

Next a construct was synthesized in which the V anchor residue in HBVPol 551 was replaced with alanine, the native residue, to address theeffect of decreasing MHC binding on epitope immunogenicity (FIG. 24B).

Unlike deletion of the Th cell epitope, decreasing the MHC bindingcapacity of the HBV Pol 551 epitope by 40-fold through modification ofthe anchor residue did not appear to affect epitope immunogenicity (FIG.25B). The CTL response against the HBV Pol 551 epitope, as well as tothe other epitopes, measured either by LU or frequency of CTL-positivecultures, was very similar between the constructs containing the nativeA or improved V residue at the MHC binding anchor site. This findingreinforces the notion that minimal epitope minigenes can efficientlydeliver epitopes of vastly different MHC binding affinities.Furthermore, this finding is particularly relevant to enhancing epitopeimmunogenicity via different delivery methods, especially in light ofthe fact that the wild type HBV Pol 551 epitope was essentiallynonimmunogenic when delivered in a less potent IFA emulsion.

Effect of the Signal Sequence on Minigene Construct Immunogenicity

The signal sequence was deleted from the pMin.1 construct, therebypreventing processing of the minigene polypeptide in the ER (FIG. 24C).When the immunogenicity of the pMin.1-No Sig construct was examined, anoverall decrease in response was found against four CTL epitopes. Two ofthese epitopes, HIV Env 120 and HBV Env 335, showed a decrease infrequency of CTL-positive cultures compared to pMin.1 while theremaining epitopes, HBV Pol 455 and HIV Pol 476, showed a 16-fold (from424 to 27 ΔLU) and 3-fold decrease (709 to 236 ΔLU) in magnitude of themean CTL response, respectively (FIG. 25C). These findings suggest thatallowing ER-processing of some of the epitopes encoded in the pMin.1prototype construct may improve immunogenicity, as compared withconstructs that allow only cytoplasmic processing of the same panel ofepitopes.

Effect of Epitope Rearrangement and Creation of New Junctional Epitopes

In the final construct tested, the immunogenicity of the HBV Env 335epitope was analyzed to determine whether it may be influenced by itsposition at the 3′ terminus of the minigene construct (FIG. 24D). Thus,the position of the Env epitope in the cDNA construct was switched witha more immunogenic epitope, HBV Pol 455, located in the center of theminigene. It should be noted that this modification also created twopotentially new epitopes. As shown in FIG. 25D, the transposition of thetwo epitopes appeared to affect the immunogenicity of not only thetransposed epitopes but also more globally of other epitopes. Switchingepitopes resulted in obliteration of CTL induction against HBV Env 335(no positive cultures detected out of six). The CTL response induced bythe terminal HBV Pol 455 epitope was also decreased but only slightly(424 vs 78 mean ΔLU). In addition to the switched epitopes, CTLinduction against other epitopes in the pMin. 1-Switch construct wasalso markedly reduced compared to the prototype construct. For example,a CTL response was not observed against the HIV Env 120 epitope and itwas significantly diminished against the HBV Core 18 (4 of 6 positivecultures, decrease in mean ΔLU from 306 to 52) and HBV Pol 476 (decreasein mean ΔLU from 709 to 20) epitopes (FIG. 25D).

As previously mentioned, it should be noted that switching the twoepitopes had created new junctional epitopes. Indeed, in thepMin.1-Switch construct, two new potential CTL epitopes were createdfrom sequences of HBV Env 335-HIV Pol 476 (LLVPFVIL (SEQ ID NO:123),H-2K^(b)-restricted) and HBV Env 335-HBV Pol 551 (VLGVWLSLLV (SEQ IDNO:136), HLA-A2.1-restricted) epitopes. Although these junctionalepitopes have not been examined to determine whether or not they areindeed immunogenic, this may account for the low immunogenicity of theHBV Env 335 and HIV Pol 476 epitopes. These findings suggest thatavoiding junctional epitopes may be important in designing multi-epitopeminigenes as is the ability to confirm their immunogenicity in vivo in abiological assay system such as HLA transgenic mice.

Induction of CTLs Against A11 Epitopes Encoded in pMin. 1

To further examine the flexibility of the minigene vaccine approach forinducing a broad CTL response against not only multiple epitopes butalso against epitopes restricted by different HLA alleles, HLA-A11K^(b)transgenic mice were immunized to determine whether the three Al Iepitopes in the pMin.1 construct were immunogenic for CTLs, as was thecase for the A2.1-restricted epitopes in the same construct. Assummarized in Table 12, significant CTL induction was observed in amajority of cultures against all three of the HLA-A11-restrictedepitopes and the level of CTL immunity induced for the three epitopes,in the range of 40 to 260 ΔLU, exceeded that of peptides delivered inIFA (Table 10). Thus,.nine CTL epitopes of varying HLA restrictionsincorporated into a prototype minigene construct all demonstratedsignificant CTL induction in vivo, confirming that minigene DNA plasmidscan serve as means of delivering multiple epitopes, of varying HLArestrictions and MHC binding affinities, to the immune system in animmunogenic fashion and that appropriate transgenic mouse strains can beused to measure DNA construct immunogenicity in vivo.

CTLs were also induced against three A11 epitopes in A11/K^(b)transgenic mice. These responses suggest that minigene delivery ofmultiple CTL epitopes that confers broad population coverage may bepossible in humans and that transgenic animals of appropriate haplotypesmay be a useful tools in optimizing the in vivo immunogenicity ofminigene DNA. In addition, animals such as monkeys having conserved HLAmolecules with cross reactivity to CTL and HTL epitopes recognized byhuman MHC molecules can be used to determine human immunogenicity of HTLand CTL epitopes (Bertoni et al., J. Immunol.161:4447-4455 (1998)).

This study represents the first description of the use of HLA transgenicmice to quantitate the in vivo immunogenicity of DNA vaccines, byexamining response to epitopes restricted by human HLA antigens. In vivostudies are required to address the variables crucial for vaccinedevelopment, that are not easily evaluated by in vitro assays, such asroute of administration, vaccine formulation, tissue biodistribution,and involvement of primary and secondary lymphoid organs. Because of itssimplicity and flexibility, HLA transgenic mice represent an attractivealternative, at least for initial vaccine development studies, comparedto more cumbersome and expensive studies in higher animal species, suchas nonhuman primates. The in vitro presentation studies described abovefurther supports the use of HLA transgenic mice for screening DNAconstructs containing human epitopes inasmuch as a direct correlationbetween in vivo immunogenicity and in vitro presentation was observed.Finally, strong CTL responses were observed against all six A 2.1restricted viral epitopes and in three A11 restricted epitopes encodedin the prototype pMin.1 construct. For five of the A 2.1 restrictedepitopes, the magnitude of CTL response approximated that observed withthe lipopeptide, Theradigm-HBV, that previously was shown to inducestrong CTL responses in humans (Vitiello et al., J. Clin. Invest. 95:341(1995); Livingston et al., J. Immunol. 159:1383 (1997)).

TABLE 1 HBV derived HTL epitopes SEQ ID Peptide Sequence Source NO:1298.06 KQAFTFSPTYKAFLC HBV POL 661 137 F107.03 LQSLTNLLSSNLSWL RBV POL412 138 1280.06 AGFFLLTRILTlPQS HBV ENV 180 139 1280.09 GTSFVYVPSALNPADHBV POL 774 140 CF-08 VSFGVWIRTPPAYRPPNAPI HBV NUC 120 141 27.0280GVWIRTPPAYRPPNA HBV NUC 123 142 1186.25 SFGVWIRTPPAYRPP HBV NUC 121 14327.0281 RHYLHTLWKAGILYK HBV POL 145 144 F107.04 PFLLAQFTSAICSVV HBV POL523 145 1186.15 LVPFVQWFVGLSPTV HBV ENV 339 146 1280.15 LHLYSHPllLGFRKlHBV POL 501 147 1298.04 KQCFRKLPVNRPIDW HBV POL 615 148 1298.07AANWILRGTSFVYVP HBV POL 764 149 857.02 PHHTALRQAILCWGELMTLA HBV CORE 50150 35.0100 LCQVFADATPTGWGL HBV POL 683 151 35.0096 ESRLVVDFSQFSRGN HBVPOL 387 152 35.0093 VGPLTVNEKRRLKLl HBV POL 96 153 1186.18NLSWLSLDVSAAFYH HBV POL 422 154

TABLE 2 HBV derived CTL epitopes Supertype Peptide Sequence Source SEQID NO: A2 924.07 FLPSDFFPSV HBV core 18-27 91 1013.0102 WLSLLVPFVHBVadr-ENV (S Ag 335-343) 107 777.03 FLLTRILTI HBV ENV ayw 183 155927.15 ALMPLYACI HBV ayw pol 642 156 1168.02 GLSRYVARL HBV POL 455 99927.11 FLLSLGIHL HBV pol 562 157 A3 1147.16 HTLWKAGILYK HBV POL 149 881083.01 STLPETTVVRR HBV core 141 103 1090.11 SAICSVVRR HBV pol 531 1581090.10 QAFTFSPTYK HBV pol 665 159 1069.16 NVSIPWTHK HBV pol 47 1601069.20 LVVDFSQFSR HBV pol 388 161 1142.05 KVGNFTGLY HBV adr POL 629 1621069.15 TLWKAGILYK HBV pol 150 163 B7 1145.04 IPIPISSWAF HBV ENV 313 164988.05 LPSDFFPSV HBV core 19-27 165 1147.04 TPARVTGGVF HBV POL 354 166A2 1069.06 LLVPFVQWFV HBV env 338-347 167 1147.13 FLLAQFTSAI HBV POL 513168 1147.14 VLLDYQGMLPV HBV ENV 259 169 1132.01 LVPFVQWFV HBV ENV 339170 1069.05 LLAQFTSAI HBV pol 504-512 171 927.42 NLSWLSLD-V HBV pol 411172 927.41 LLSSNLSWL HBV pol 992 173 927.46 KLHLYSHPI HBV pol 489 1741069.071 FLLAQFRSA HBV pol 503 175 1142.07 GLLGWSPQA HBV ENV 62 176927.47 HLYSHPIIL HBV ayw pol 1076 177 1069.13 PLLPIFFCL HBV env 377-385178 103.1402 VLQAGFFLL HBVadr-ENV 177 179 1090.14 YMDDVVLGA HBV pol538-546 95 A3 26.0539 RLVVDFSQFSR HBV pol 376 180 26.0535 GVWIRTPPAYRHBV X niuc fus 299 181 A3 26.0153 SSAGPCALR HBV X 64 182 1.0993KVFVLGGCR HBV adr “X” 1548 183 26.0149 CALRFTSAR HBV X 69 184 26.0023VSFGVWIR HBV x nuc fus 296 185 26.0545 TLPETUVVRRR HBV x nuc fus 318 18620.0131 SVVRRAFPH HBV POL 524 187 1.0219 FVLGGCRHK HBV adr “X” 1550 18826.0008 FTFSPTYK HBV pol 656 189 20.0130 AFTESPTYK HBV POL 655 190 B71147.05 FPHCLAFSYM HBV POL 530 191 1147.08 YPALMPLYA HBV POL 640 1921147.06 LPVCAFSSA HBV X 58 193 1147.02 HPAAMPHLL HBV POL 429 194 26.0570YPALMPLYACI HBV pol 640 195 19.0014 YPALMPLY HBV POL 640 196 1145.08FPHCLAFSY HBV POL 541 197 Other 1090.02 AYRPPNAPI HBV NUC 131 198 1.0519DLLDTASALY HBV adr CORE 419 199 13.0129 EYLVSFGVWI HBV NUC 117 20020.0254 FAAPFTQCGY HBV POL 631 201 2.0060 GYPALMPLY HBV ALL 1224 2021069.04 HTLWKAGILY HBV pol 149 203 1069.08 ILLLCLIFLL HBV env 249-258204 1.0166 KVGNFTGLY HBV adr POL 629 162 1069.23 KYTSFPWLL HBV POL 745205 1069.01 LLDTASALY HBV core 59 26 2.0239 LSLDVSAAFY HBV ALL 1000 2072.0181 LYSRPIILGF HBV POL 492 208 1039.01 MMWYWGPSLY HBV 360 209 2.0126MSTTDLEAY HBV adr 1521 210 1069.03 PLDKGIKPYY HBV pol 124 211 1090.09PTTGRTSLY HBV pol 808 212 20.0138 PWTHKVGNF HBV POL 51 213 20.0135RWMCLRRFI HBV ENV 236 214 20.0269 RWMCLRRFII HBV ENV 236 215 20.0139SFCGSPYSW HBV POL 167 216 Other 1069.02 SLDVSAAFY RBV pol 427 21720.0136 SWLSLLVPF HBV ENV 334 218 20.0271 SWPKFAVPNL HBV POL 392 21920.0137 SWWTSLNFL HBV ENV 197 220 2.0173 SYQHFRKLLL HBV POL 4 22113.0073 WFHISCLTF HBV NUC 102 222 1.0774 WLWGMDIDPY HBV adw CORE 416 2231039.06 WMMWYWGPSLY HBV env 359 224 924.14 FLPSDFFPSI HBv 18-27 I₁₀ var.225 1090.77 YMDDVVLGV HBV pol 538-546 sub 462 941.01 FLPSDYFPSV HBc18-27 analog 226 1083.02 STLPETYVVRR HBV core 141-151 analog 227 1145.05FPIPSSWAF HBV ENV 313 analog 228 1145.11 FPHCLAFSL HBV POL 541 analog229 1145.24 FPHCLAFAL HBV POL 541 analog 230 1145.06 IPITSSWAF HBV ENV313 analog 231 145.23 IPIPMSWAF HBV ENV 313 analog 232 1145.07 IPILSSWAFHBV ENV 313 analog 233 1145.09 FPVCLAFSY HBV POL 541 analog 234 1145.10FPHCLAFAY HBV POL 541 analog 235

TABLE 3 HCV derived HTL epitopes Peptide Sequence Source SEQ ID NO:AAYAAQGYKVLVLNPSVAATLGFGAY HCV NS3 1242-1267 236 P98.03AAYAAQGYKVLVLNPSVAAT HCV NS3 1242 237 P98.04 GYKVLVLNPSVAATLGFGAY HCVNS3 1248 238 P98.05 GYKVLVLNPSVAAT HCV NS3 1248 239 1283.21GYKVLVLNPSVAATL HCV NS3 1253 240 1283.20 AQGYKVLVLNPSVAA HCV NS3 1251241 GEGAVQWMNRLIAFASRGNHVS HCV NS4 1914-1935 242 F134.08GEGAVQWMNRLIAFASRGNHV HCV NS4 1914 243 1283.44 MNRLIAFASRGNHVS HCV NS41921 244 1283.16 SKGWRLLAPITAYAQ HCV NS3 1025 245 1283.55GSSYGFQYSPGQRVE HCV NS5 2641 246 F134.05 NFISGIQYLAGLSTLPGNPA HCV NS41772 247 1283.61 ASCLRKLGVPPLRVW HCV NS5 2939 248 1283.25GRHLIFCHSKKKCDE HCV NS3 1393 249 35.0107 TVDFSLDPTFTIETT HCV 1466 25035.0106 VVVVATDALMTGYTG HCV 1437 251

TABLE 4 HCV derived CTL epitopes Super- SEQ type Peptide Sequence SourceID NO: A2 1090.18 FLLLADARV HCV NS I/E2 728 252 1073.05 LLFNILGGWV HCVNS4 1812 253 1013.02 YLVAYQATV HCV NS3 1590 254 1013.1002 DLMGYIPLV HCVCore 132 255 1090.22 RLIVFPDLGV HCV NS5 2611 256 24.0075 VLVGGVLAA HCVNS4 1666 257 24.0073 WMNRLIAFA HCV NS4 1920 258 1174.08 HMWNFISGI HCVNS4 1769 259 1073.06 ILAGYGAGV HCV NS4 1851 260 24.0071 LLFLLLADA HCVNS1/E2 726 261 1073.07 YLLPRRGPRL HCV Core 35 262 1.0119 YLVTRHADV HCVNS3 1136 263 A3 1.0952 KTSERSQPR HCV Core 51 264 1073.10 GVAGALVAFR HCVNS4 1863 265 1.0123 LIFCHSKKK HCV NS3 1391 266 1.0955 QLFTFSPRR HCV E1290 267 1073.11 RLGVRATRK HCV Core 43 268 1073.13 RMYVGGVEHR HCV NS1/E2635 269 24.0090 VAGALVAFK HCV NS4 1864 270 F104.01 VGIYLLPNR HCV NS53036 271 B7 1145.12 LPGCSFSIF HCV Core 168 272 29.0035 IPFYGKAI HCV 1378273 Other 1069.62 CTCGSSDLY HCV NS3 1128 274 24.0092 FWAKHMVNF HCV NS41765 275 13.0019 LSAFSLRSY HCV NS5 2922 276 A3 24.0086 LGFGAYMSK HCV NS31267 277 1174.21 RVCEKMALY HCV NS5 2621 278 1174.16 WMNSTGFTK HCV NS1/E2557 279 1073.04 TLHGPTPLLY HCV NS3 1622 280 B7 16.0012 FPYLVAYQA HCV NS31588 281 15.0047 YPCTVNFTI HCV NS1/E2 623 282 Other 24.0093 EVDGVRLHRYHCV NS5 2129 283 3.0417 LTCGFADLMGY HCV 126 284 1073.01 NIVDVQYLY HCV E1700 285 1.0509 GLSAFSLHSY HCV NS5 2921 286 1073.17 MYVGDLCGSVF HCV E1275 287 1073.18 MYVGGVEHRL HCV NS1/E2 633 288 13.075 QYLAGLSTL HCV NS41778 289 1145.13 FPGCSFSIF HCV Core 168 290 1145.25 LPGCMFSIF HCV Core168 291 1292.24 LPGCSFSII HCV Core 169 292 1145.14 LPVCSFSIF HCV Core168 293 1145.15 LPGCSFSYF HCV Core 168 294

TABLE 5 HIV derived HTL epitopes Peptide Sequence Source SEQ ID NO:GEIYKRWIILGLNKIVRMYSPTSILD HIV1 GAG 294-319 295 KRWIILGLNKIVRMYSPTSILDHIV gag 298-319 296 27.0313 KRWIILGLNKIVRMY HIV1 GAG 298 297 27.0311GEIYKRWIILGLNKI HIV1 GAG 294 298 27.0354 WEFVNTPPLVKLWYQ HIV1 POL 596299 27.0377 QKQITKIQNFRVYYR HIV1 POL 956 300 EKVYLAWVPAHKGIGG HIV1 POL711-726 301 1280.03 KVYLAWVPAHKGIGG HIV POL 712 302 27.0361EKVYLAWVPAHKGIG HIV1 POL 711 303 PIVQNIQGQMVHQAISPRTLNA HIV1 gag 165-186304 27.0304 QGQMVHQAISPRTLN HIV1 GAG 171 305 27.0297 QHLLQLTVWGIKQLQHIV1 ENV 729 306 27.0344 SPAIFQSSMTKILEP HIV1 POL 335 307 F091.15IKQFINMWQEVGKAMY HIV1 ENV 566 308 27.0341 FRKYTAFTIPSINNE HIV1 POL 303309 27.0364 HSNWRAMASDFNLPP HIV1 POL 758 310 27.0373 KTAVQMAVFIHNFKRHIV1 POL 915 311 DRVHPVHAGPIAPGQMREPRGS HIV GAG 245 312AFSPEVIPMFSALSEGATPQDLNTML HIV gag 195-216 313 AFSPEVIPMFSALSEGATPQDLHIV gag 195-216 314 200.06 SALSEGATPQDLNIMLT HIV gag 205 315 27.0307SPEVIPMFSALSEGA HIV gag 197 316 LQEQIGWMTNNPPIPVGEIYKR HIV gag 275 31727.0310 QEQIGWMTNNPPIPV HIV gag 276 3218  35.0135 YRKILRQRKIDRLID HIVVPU 31 319 35.0131 WAGIKQEFGIPYNPQ HIV POL 874 320 35.0127EVNIVTDSQYALGII HIV POL 674 321 35.0125 AETFYVDGAANRETK HIV POL 619 32235.0133 GAVVIQDNSDIKVVP HIV POL 989 323

TABLE 6 HIV derived CTL epitopes Supertype Peptide Sequence Source SEQID NO: A2 25.0148 MASDFNLPPV HIV1 POL 70 324 1069.32 VLAEAMSQV HIV gag397 325 1211.04 KLTPLCVTL HIV ENV 134 326 25.0062 KILVGKLNWA HIV1 POL 87463 25.0039 LTFGWCFKL HIV1 NEF 62 327 941.031 ILKEPVHGV HIVI pol 476-484101 25.0035 MTNNPPIPV HIV1 GAG 34 328 25.0057 RILQQLLFI HIV1 VPR 72 329A3 1.0944 AVFIHNFKR HIV POL 1434 330 1.1056 KIQNFRVYYR HIV POL 1474 3311069.49 QMAVFIHNFK HIV pol 1432 332 966.0102 AIFQSSMTK HIV pol 337 3331150.14 MAVFIHNFK HIV pol 909 334 940.03 QVPLRPMTYK HIV nef 73-82 33525.0175 TTLFCASDAK HIV1 ENV 81 336 1069.43 TVYYGVPVWK HIV env 49 10525.0209 VTIKIGGQLK HIV1 POL 65 337 B7 1146.01 FPVRPQVPL HIV nef 84-92338 29.0060 IPIHYCAPA HIV env 293 339 15.0073 FPISPIETV HIV POL 171 34029.0056 CPKVSFEPI HIV env 285 341 29.0107 IPYNPQSQGVV HIV pol 883 342 A225.0151 CTLNFPISPI HIV1 POL 96 343 25.0143 LTPGWCFKLV HIV1 NEFP 62 34425.0043 YTAFTIPSI HIV1 POL 83 345 25.0055 AIIRILQQL HIV1 VPR 76 34625.0049 ALVEICTEM HIV1 POL 52 347 25.0032 LLQLTVWGI HIV1 ENV 61 34825.0050 LVGPTPVNI HIV1 POL 100 349 25.0047 KAACWWAGI HIV1 POL 65 35025.0162 KMIGGIGGFI HIV1 POL 96 351 25.0052 RAMASDFNL HIV1 POL 78 3521211.09 SLLNATDIAV HIV ENV 814 353 A2 25.0041 TLNFPISPI HIV1 POL 96 354A3 1.0046 IVIWGKTPK HIV POL 1075 355 25.0064 MVHQAISPR HIV1 GAG 45 3561.0062 YLAWVPAHK HIV POL 1227 357 1.0942 MTKILEPFR HIV POL 859 35825.0184 QMVHQAISPR HIV1 GAG 45 359 1069.48 AVFIHNFKRK HIV pol 1434 3601069.44 KLAGRWPVK HIV pol 1358 361 1069.42 KVYLAWVPAHK HIV pol 1225 3621.0024 NTPVFAIKK HIV pol 752 363 25.0062 RIVELLGRR HIV1 ENV 53 36425.0095 TIKIGGQLK HIV1 POL 65 365 25.0078 TLFCASDAK HIV1 ENV 82 36625.0104 VMIVWQVDR HIV1 VIF 83 367 1069.47 VTVYYGVPVWK HIV env 48 368 B715.0268 YPLASLRSLF HIV GAG 507 369 1292.13 HPVHAGPIA HIV GAG 248 37019.0044 VPLQLPPL HJV con. REV 71 371 Other 1.0431 EVNIVTDSQY HIV POL1187 372 1.0014 FRDYVDRFY HIV GAG 298 373 25.0113 IWGCSGKLI HIV1 ENV 69374 25.0127 IYETYGDTW HIV1 VPR 92 375 1069.60 IYQEPFKNL HIV pol 1036 3762.0129 IYQYMDDLY HIV pol 359 377 25.0128 PYNEWTLEL HIV1 VPR 56 37825.0123 PYNTPVFAI HIV1 POL 74 379 1069.57 RYLKDQQLL HIV env 2778 3801069.58 RYLRDQQLL HIV env 2778 381 1069.59 TYQIYQEPF HIV pol 1033 3821069.27 VIYQYMDDLY HIV pol 358 383 1069.26 VTVLDVGDAY HIV pol 265 38425.0115 VWKEATTTL HIV1 ENV 47 385 25.0218 VWYEATITLF HIV1 ENV 47 38625.0219 YMQATWIPEW HIV1 POL 96 387 A2 1211.4 SLLNATAIAV HIV MN gp 160814(a) 388 A3 F105.21 AIFQRSMTR HIV pol 337(a) 389 F105.17 AIFQSSMTR HIVpol 337(a) 390 F105.02 GIFQSSMTY HIV pol 337(a) 391 F105.03 AAFQSSMTKHIV pol 337(a) 392 F105.04 AIAQSSMTK HIV pol 337(a) 393 F105.05AIFASSMTK HIV pol 337(a) 394 F105.06 AIFQASMTK HIV pol 337(a) 395F105.07 AIFQSAMTK HIV pol 337(a) 396 F105.08 AIFQSSATK HIV pol 337(a)397 F105.09 AIFQSSMAK HIV pol 337(a) 398 F105.11 FIFQSSMTK HIV pol337(a) 399 F105.12 SIFQSSMTK HIV pol 337(a) 400 F105.16 AIFQCSMTK HIVpol 337(a) 401 B7 1145.03 FPVRPQFPL HIV nef 84-92 analog 402 1181.03FPVRPQVPI HIV nef 84-92(a) 403 1292.14 HPVHAGPII HIV GAG 248 404 1292.09FPISPIETI HIV POL 179 405 1145.02 FPVFQVPL HIV nef 84-92 analog 4061145.22 TPVRMQVPL HIV nef 84-92 analog 407 1181.04 FPVRPQVPM HIV nef84-92(a) 408 1181.01 FPVRPQVPA HIV nef 84-92(a) 409 1181.02 FPVRPQVPVHIV nef 84-92(a) 410 1181.05 FPVRPQVPF HIV nef 84-92(a) 411 1181.06FPVRPQVPW HIV nef 84-92(a) 412

TABLE 7 P. falciparum derived HTL epitopes Peptide Sequence Source SEQID NO: F125.04 RHNWVNHAVPLAMKLI Pf SSP2 61 473 1188.34 HNWVNHAVPLAMKLIPf SSP2 62 414 1188.16 KSKYKLATSVLAGLL Pf EXP1 71 415 LVNLLIFHINGKIIKNSEPf LSA1 13 416 F125.02 LVNLLIFHINGKIIKNS Pf LSA1 13 417 27.0402LLIFHINGKIIKNSE Pf LSA1 16 418 1188.32 GLAYKFVVPGAATPY Pf SSP2 512 41927.0392 SSVFNVVNSSIGLIM Pf CSP 410 420 27.0417 VKNVTGPFMKAVCVE Pf SSP2223 421 27.0388 MRKLAILSVSSFLFV Pf CSP 2 422 27.0387 MNYYGKQENWYSLKK PfCSP 53 423 1188.38 KYKIAGGIAGGLALL Pf SSP2 494 424 1188.13AGLLGNVSTVLLGGV Pf EXP1 82 425 27.0408 QTNFKSLLRNLGVSE Pf LSA1 94 42635.0171 PDSIQDSLKESRKLN Pf SSP2 165 427 35.0172 KCNLYADSAWENVKN Pf SSP2211 428

TABLE 8 P. falciparum derived CTL epitopes Supertype Peptide SequenceSource SEQ ID NO: A2 1167.21 FLIFFDLFLV Pf SSP2 14 429 1167.08 GLIMVLSFLPf CSP 425 430 1167.12 VLAGLLGNV Pf EXP1 80 431 1167.13 KILSVFFLA PfEXP1 2 432 1167.10 GLLGNVSTV Pf EXP183 433 1167.18 ILSVSSFLFV Pf CSP 7434 1167.19 VLLGGVGLVL Pf EXP 1 91 435 A3 1167.36 LACAGLAYK Pf SSP2 511436 1167.32 QTNFKSLLR Pf LSA1 94 437 1167.43 VTCGNGIQVR Pf CSP 375 4381167.24 ALFFIINK Pf EXP1 10 439 167.28 GVSENIFLK Pf LSA 1 105 4401167.47 HVLSHNSYEK Pf LSA1 59 441 1167.51 LLACAGLAYK Pf SSP2 510 4421167.46 FILVNLLIFH Pf LSA1 11 443 B7 1101.03 MPLETQLAI Pf SHEBA 77 4441167.61 TPYAGEPAPF Pf SSP2 539 445 A2 1167.14 FLIFFDLFL Pf SSP2 14 4461167.16 FMKAVCVEV Pf SSP2 230 447 1167.15 LIFFDLFLV Pf SSP2 15 4481167.17 LLMDCSGSI Pf SSP2 51 449 1167.09 VLLGGVGLV Pf EXP1 91 450 B719.0051 LPYGRTNL Pf SSP2 126 451 Other 16.0245 FQDEENIGIY Pf LSA1 1794452 16.0040 FVEALFQEY Pf CSP 15 453 1167.54 FYFTLVNLL Pf LSA1 9 4541167.53 KYKLATSVL Pf EXP1 73 455 1167.56 KYLVIVFLI Pf SSP2 8 456 15.0184LPSENERGY Pf LSA1 1663 457 16.0130 PSDGKCNLY Pf SSP2 207 458 16.0077PSENERGYY Pf LSA 1 1664 459 1167.57 PYAGEPAPF Pf SSP2 528 460 1167.55YYIPHQSSL Pf LSA1 1671 461

TABLE 9 Activation of T Cell Proliferation by Expression VectorsEncoding MHC Class II Epitopes Fused to MHC Class II Targeting SequencesStimulating Peptide¹ Immunogen PADRE OVA 323 CORE 128 peptide + CFA² 3.0(1.1) 2.7 (1.2) 3.2 (1.4) pEP2.(PAOS).(-) — — — pEP2.(AOS).(-) 5.6 (1.8)— — pEP2.(PAOS).(sigTh) 5.0 (2.9) — 2.6 (1.5) pEP2.(PAOS).(IgαTh) 5.6(2.1) — 3.0 (1.6) pEP2.(PAOS).(LampTh) 3.8 (1.7) — 3 pEP2.(PAOS).(IiTh)5.2 (2.0) 3.2 (1.5) 3.7 (1.5) pEP2.(PAOS).(H2M) 3.3 (1.3) — 2.8¹Geometric mean of cultures with SI ≧ 2. ²Proliferative responsemeasured in the lymph node.

TABLE 10 CTL Epitopes in CDNA Minigene MHC Immunogenicity In Vivo (IFA)Binding No. CTL- CTL Response MHC Affinity Positive (Geo. Mean EpitopeSequence Restrict. [IC30% Cultures ΔLU SEQ ID NO: HBV Core 18 FLPSDFFPSVWLSLLVPFV A2.1 (nM) 6/6 73.0 (1.1) 91 HBV Env 335 GLSRYVARL KLTPLCVTLA2.1 3 4/6  5.3 (1.6) 107 HBV Pol 455 ILKEPVRGV YMDDVVLGA A2.1 5 ND^(c)ND 99 HIV Env 120 YMDDVVLGV A2.1 76 2/5  6.4 (1.3) 93 HIV Pol 476TVYYGVPVWK A2.1 102 2/5 15.2 (2.9) 101 HBV Pol 55 1-A STLPETRVVRR A2.1192 0/6 — 95 HBV Pol 55 1-V HTLWKAGILYK A2.1 200 6/6  8.2 (2.3) — HIVEnv 49 A11 5 28/33 13.4 (3.1) 105 HBV Core 141 A11 4 6/6 12.1 (2.6) 103HBV Pol 149 A11 4 6/6 13.1 (1.2) 14 8 ^(a)Peptide tested inHLA-A2.1/K^(b)H-2^(bxs) transgenic mice by co-immunizing with a T helpercell peptide in IFA. ^(b)Geometric mean CTL response of positivecultures. ^(c)ND, not done.

TABLE 11 Summary of Immunogenicity of pMin.1 DNA construct in HLAA2.l/K^(b) transgenic mice CTL Response^(a) Geo. Mean Response PositiveNo. Positive Cultures [×/÷ SD] Epitope Cultures/Total^(b) ΔLU HBV Core18 9/9 455.5 [2.2] HIV Env 120 12/12 211.9 [3.7] HBV Pol 551-V 9/9 126.1[2.8] HBV Pol 455 12/12 738.6 [1.3] HIV Pol 476 11/11 716.7 [1.5] HBVEnv 335 12/12  43.7 [1.8] HBV Core 18 10/10 349.3 [1.8] (Theradigm)^(c)^(a)Mice were immunized with pMin.1 DNA or Theradigm-HBV lipopeptide andCTL activity in splenocyte cultures was determined after in vitrostimulation with individual peptide epitopes. Results from fourindependent experiments are shown. ^(b)See Example V, Materials andMethods for definition of a CTL-positive culture. ^(c)Response of miceimmunized with Theradigm-HBV lipopeptide containing the HBV Core 18epitope.

TABLE 12 Summary of immunogenicity in HLA A11/K^(b) transgenic mice CTLResponse^(a) Geo. Mean Response No. Positive Positive Cultures [×/÷ SD]Epitope Cultures/Total^(b) ΔLU HBV Core 141 5/9 128.1 [1.6] HBV Pol 1496/9 267.1 [2.2] HIV Env 43 9/9  40.1 [2.9] ^(a)Mice were immunized withpMin.1 DNA and CTL activity in splenocyte cultures was determined afterin vitro stimulation with individual A11-restricted epitopes. Thegeometric mean CTL response from three independent experiments areshown. ^(b)Definition of a CTL-positive culture is described in ExampleV, Materials and Methods.

463 1 669 DNA Artificial Sequence IiPADRE construct encoding fusion ofmurine Ii gene with pan DR epitope sequences substituted for CLIPsequence 1 gctagcgccg ccacc atg gat gac caa cgc gac ctc atc tct aac catgag 51 Met Asp Asp Gln Arg Asp Leu Ile Ser Asn His Glu 1 5 10 caa ttgccc ata ctg ggc aac cgc cct aga gag cca gaa agg tgc agc 99 Gln Leu ProIle Leu Gly Asn Arg Pro Arg Glu Pro Glu Arg Cys Ser 15 20 25 cgt gga gctctg tac acc ggt gtt tct gtc ctg gtg gct ctg ctc ttg 147 Arg Gly Ala LeuTyr Thr Gly Val Ser Val Leu Val Ala Leu Leu Leu 30 35 40 gct ggg cag gccacc act gct tac ttc ctg tac cag caa cag ggc cgc 195 Ala Gly Gln Ala ThrThr Ala Tyr Phe Leu Tyr Gln Gln Gln Gly Arg 45 50 55 60 cta gac aag ctgacc atc acc tcc cag aac ctg caa ctg gag agc ctt 243 Leu Asp Lys Leu ThrIle Thr Ser Gln Asn Leu Gln Leu Glu Ser Leu 65 70 75 cgc atg aag ctt ccgaaa tct gcc aaa cct gtg gcc aag ttc gtg gct 291 Arg Met Lys Leu Pro LysSer Ala Lys Pro Val Ala Lys Phe Val Ala 80 85 90 gcc tgg acc ctg aag gctgcc gct atg tcc atg gat aac atg ctc ctt 339 Ala Trp Thr Leu Lys Ala AlaAla Met Ser Met Asp Asn Met Leu Leu 95 100 105 ggg cct gtg aag aac gttacc aag tac ggc aac atg acc cag gac cat 387 Gly Pro Val Lys Asn Val ThrLys Tyr Gly Asn Met Thr Gln Asp His 110 115 120 gtg atg cat ctg ctc acgagg tct gga ccc ctg gag tac ccg cag ctg 435 Val Met His Leu Leu Thr ArgSer Gly Pro Leu Glu Tyr Pro Gln Leu 125 130 135 140 aag ggg acc ttc ccagag aat ctg aag cat ctt aag aac tcc atg gat 483 Lys Gly Thr Phe Pro GluAsn Leu Lys His Leu Lys Asn Ser Met Asp 145 150 155 ggc gtg aac tgg aagatc ttc gag agc tgg atg aag cag tgg ctc ttg 531 Gly Val Asn Trp Lys IlePhe Glu Ser Trp Met Lys Gln Trp Leu Leu 160 165 170 ttt gag atg agc aagaac tcc ctg gag gag aag aag ccc acc gag gct 579 Phe Glu Met Ser Lys AsnSer Leu Glu Glu Lys Lys Pro Thr Glu Ala 175 180 185 cca cct aaa gag ccactg gac atg gaa gac cta tct tct ggc ctg gga 627 Pro Pro Lys Glu Pro LeuAsp Met Glu Asp Leu Ser Ser Gly Leu Gly 190 195 200 gtg acc agg cag gaactg ggt caa gtc acc ctg tgaggtacc 669 Val Thr Arg Gln Glu Leu Gly GlnVal Thr Leu 205 210 215 2 215 PRT Artificial Sequence IiPADRE 2 Met AspAsp Gln Arg Asp Leu Ile Ser Asn His Glu Gln Leu Pro Ile 1 5 10 15 LeuGly Asn Arg Pro Arg Glu Pro Glu Arg Cys Ser Arg Gly Ala Leu 20 25 30 TyrThr Gly Val Ser Val Leu Val Ala Leu Leu Leu Ala Gly Gln Ala 35 40 45 ThrThr Ala Tyr Phe Leu Tyr Gln Gln Gln Gly Arg Leu Asp Lys Leu 50 55 60 ThrIle Thr Ser Gln Asn Leu Gln Leu Glu Ser Leu Arg Met Lys Leu 65 70 75 80Pro Lys Ser Ala Lys Pro Val Ala Lys Phe Val Ala Ala Trp Thr Leu 85 90 95Lys Ala Ala Ala Met Ser Met Asp Asn Met Leu Leu Gly Pro Val Lys 100 105110 Asn Val Thr Lys Tyr Gly Asn Met Thr Gln Asp His Val Met His Leu 115120 125 Leu Thr Arg Ser Gly Pro Leu Glu Tyr Pro Gln Leu Lys Gly Thr Phe130 135 140 Pro Glu Asn Leu Lys His Leu Lys Asn Ser Met Asp Gly Val AsnTrp 145 150 155 160 Lys Ile Phe Glu Ser Trp Met Lys Gln Trp Leu Leu PheGlu Met Ser 165 170 175 Lys Asn Ser Leu Glu Glu Lys Lys Pro Thr Glu AlaPro Pro Lys Glu 180 185 190 Pro Leu Asp Met Glu Asp Leu Ser Ser Gly LeuGly Val Thr Arg Gln 195 200 205 Glu Leu Gly Gln Val Thr Leu 210 215 3438 DNA Artificial Sequence I80T construct encoding fusion of thecytoplasmic, transmembrane and part of the luminal domains of murine Iiprotein gene fused to multiple MHC class II epitopes 3 gctagcgccg ccaccatg gat gac caa cgc gac ctc atc tct aac cat gag 51 Met Asp Asp Gln ArgAsp Leu Ile Ser Asn His Glu 1 5 10 caa ttg ccc ata ctg ggc aac cgc cctaga gag cca gaa agg tgc agc 99 Gln Leu Pro Ile Leu Gly Asn Arg Pro ArgGlu Pro Glu Arg Cys Ser 15 20 25 cgt gga gct ctg tac acc ggt gtt tct gtcctg gtg gct ctg ctc ttg 147 Arg Gly Ala Leu Tyr Thr Gly Val Ser Val LeuVal Ala Leu Leu Leu 30 35 40 gct ggg cag gcc acc act gct tac ttc ctg taccag caa cag ggc cgc 195 Ala Gly Gln Ala Thr Thr Ala Tyr Phe Leu Tyr GlnGln Gln Gly Arg 45 50 55 60 cta gac aag ctg acc atc acc tcc cag aac ctgcaa ctg gag agc ctt 243 Leu Asp Lys Leu Thr Ile Thr Ser Gln Asn Leu GlnLeu Glu Ser Leu 65 70 75 cgc atg aag ctt atc agc cag gct gtg cac gcc gctcac gcc gaa atc 291 Arg Met Lys Leu Ile Ser Gln Ala Val His Ala Ala HisAla Glu Ile 80 85 90 aac gaa gct gga aga acc cct cca gct tat cgc cct ccaaac gct cct 339 Asn Glu Ala Gly Arg Thr Pro Pro Ala Tyr Arg Pro Pro AsnAla Pro 95 100 105 atc ctg ttc ttt ctg ctg acc aga atc ctg aca atc ccccag tcc ctg 387 Ile Leu Phe Phe Leu Leu Thr Arg Ile Leu Thr Ile Pro GlnSer Leu 110 115 120 gac gcc aag ttc gtg gct gcc tgg acc ctg aag gct gccgct 429 Asp Ala Lys Phe Val Ala Ala Trp Thr Leu Lys Ala Ala Ala 125 130135 tgaggtacc 438 4 138 PRT Artificial Sequence I80T 4 Met Asp Asp GlnArg Asp Leu Ile Ser Asn His Glu Gln Leu Pro Ile 1 5 10 15 Leu Gly AsnArg Pro Arg Glu Pro Glu Arg Cys Ser Arg Gly Ala Leu 20 25 30 Tyr Thr GlyVal Ser Val Leu Val Ala Leu Leu Leu Ala Gly Gln Ala 35 40 45 Thr Thr AlaTyr Phe Leu Tyr Gln Gln Gln Gly Arg Leu Asp Lys Leu 50 55 60 Thr Ile ThrSer Gln Asn Leu Gln Leu Glu Ser Leu Arg Met Lys Leu 65 70 75 80 Ile SerGln Ala Val His Ala Ala His Ala Glu Ile Asn Glu Ala Gly 85 90 95 Arg ThrPro Pro Ala Tyr Arg Pro Pro Asn Ala Pro Ile Leu Phe Phe 100 105 110 LeuLeu Thr Arg Ile Leu Thr Ile Pro Gln Ser Leu Asp Ala Lys Phe 115 120 125Val Ala Ala Trp Thr Leu Lys Ala Ala Ala 130 135 5 783 DNA ArtificialSequence IiThfull construct encoding fusion of the cytoplasmic,transmembrane and part of the luminal domains of the murine Ii proteingene fused to multiple T helper epitopes and amino acid residues 101-215trimerization region of the Ii protein 5 gctagcgccg ccacc atg gat gaccaa cgc gac ctc atc tct aac cat gag 51 Met Asp Asp Gln Arg Asp Leu IleSer Asn His Glu 1 5 10 caa ttg ccc ata ctg ggc aac cgc cct aga gag ccagaa agg tgc agc 99 Gln Leu Pro Ile Leu Gly Asn Arg Pro Arg Glu Pro GluArg Cys Ser 15 20 25 cgt gga gct ctg tac acc ggt gtt tct gtc ctg gtg gctctg ctc ttg 147 Arg Gly Ala Leu Tyr Thr Gly Val Ser Val Leu Val Ala LeuLeu Leu 30 35 40 gct ggg cag gcc acc act gct tac ttc ctg tac cag caa cagggc cgc 195 Ala Gly Gln Ala Thr Thr Ala Tyr Phe Leu Tyr Gln Gln Gln GlyArg 45 50 55 60 cta gac aag ctg acc atc acc tcc cag aac ctg caa ctg gagagc ctt 243 Leu Asp Lys Leu Thr Ile Thr Ser Gln Asn Leu Gln Leu Glu SerLeu 65 70 75 cgc atg aag ctt atc agc cag gct gtg cac gcc gct cac gcc gaaatc 291 Arg Met Lys Leu Ile Ser Gln Ala Val His Ala Ala His Ala Glu Ile80 85 90 aac gaa gct gga aga acc cct cca gct tat cgc cct cca aac gct cct339 Asn Glu Ala Gly Arg Thr Pro Pro Ala Tyr Arg Pro Pro Asn Ala Pro 95100 105 atc ctg ttc ttt ctg ctg acc aga atc ctg aca atc ccc cag tcc ctg387 Ile Leu Phe Phe Leu Leu Thr Arg Ile Leu Thr Ile Pro Gln Ser Leu 110115 120 gac gcc aag ttc gtg gct gcc tgg acc ctg aag gct gcc gct atg tcc435 Asp Ala Lys Phe Val Ala Ala Trp Thr Leu Lys Ala Ala Ala Met Ser 125130 135 140 atg gat aac atg ctc ctt ggg cct gtg aag aac gtt acc aag tacggc 483 Met Asp Asn Met Leu Leu Gly Pro Val Lys Asn Val Thr Lys Tyr Gly145 150 155 aac atg acc cag gac cat gtg atg cat ctg ctc acg agg tct ggaccc 531 Asn Met Thr Gln Asp His Val Met His Leu Leu Thr Arg Ser Gly Pro160 165 170 ctg gag tac ccg cag ctg aag ggg acc ttc cca gag aat ctg aagcat 579 Leu Glu Tyr Pro Gln Leu Lys Gly Thr Phe Pro Glu Asn Leu Lys His175 180 185 ctt aag aac tcc atg gat ggc gtg aac tgg aag atc ttc gag agctgg 627 Leu Lys Asn Ser Met Asp Gly Val Asn Trp Lys Ile Phe Glu Ser Trp190 195 200 atg aag cag tgg ctc ttg ttt gag atg agc aag aac tcc ctg gaggag 675 Met Lys Gln Trp Leu Leu Phe Glu Met Ser Lys Asn Ser Leu Glu Glu205 210 215 220 aag aag ccc acc gag gct cca cct aaa gag cca ctg gac atggaa gac 723 Lys Lys Pro Thr Glu Ala Pro Pro Lys Glu Pro Leu Asp Met GluAsp 225 230 235 cta tct tct ggc ctg gga gtg acc agg cag gaa ctg ggt caagtc acc 771 Leu Ser Ser Gly Leu Gly Val Thr Arg Gln Glu Leu Gly Gln ValThr 240 245 250 ctg tgaggtacc 783 Leu 6 253 PRT Artificial SequenceIiThfull 6 Met Asp Asp Gln Arg Asp Leu Ile Ser Asn His Glu Gln Leu ProIle 1 5 10 15 Leu Gly Asn Arg Pro Arg Glu Pro Glu Arg Cys Ser Arg GlyAla Leu 20 25 30 Tyr Thr Gly Val Ser Val Leu Val Ala Leu Leu Leu Ala GlyGln Ala 35 40 45 Thr Thr Ala Tyr Phe Leu Tyr Gln Gln Gln Gly Arg Leu AspLys Leu 50 55 60 Thr Ile Thr Ser Gln Asn Leu Gln Leu Glu Ser Leu Arg MetLys Leu 65 70 75 80 Ile Ser Gln Ala Val His Ala Ala His Ala Glu Ile AsnGlu Ala Gly 85 90 95 Arg Thr Pro Pro Ala Tyr Arg Pro Pro Asn Ala Pro IleLeu Phe Phe 100 105 110 Leu Leu Thr Arg Ile Leu Thr Ile Pro Gln Ser LeuAsp Ala Lys Phe 115 120 125 Val Ala Ala Trp Thr Leu Lys Ala Ala Ala MetSer Met Asp Asn Met 130 135 140 Leu Leu Gly Pro Val Lys Asn Val Thr LysTyr Gly Asn Met Thr Gln 145 150 155 160 Asp His Val Met His Leu Leu ThrArg Ser Gly Pro Leu Glu Tyr Pro 165 170 175 Gln Leu Lys Gly Thr Phe ProGlu Asn Leu Lys His Leu Lys Asn Ser 180 185 190 Met Asp Gly Val Asn TrpLys Ile Phe Glu Ser Trp Met Lys Gln Trp 195 200 205 Leu Leu Phe Glu MetSer Lys Asn Ser Leu Glu Glu Lys Lys Pro Thr 210 215 220 Glu Ala Pro ProLys Glu Pro Leu Asp Met Glu Asp Leu Ser Ser Gly 225 230 235 240 Leu GlyVal Thr Arg Gln Glu Leu Gly Gln Val Thr Leu 245 250 7 378 DNA ArtificialSequence KappaLAMP-Th construct encoding fusion of murine immunoglobulinkappa signal sequence fused to multiple T helper epitopes and thecytoplasmic and transmembrane domains of human lysosomal membraneglycoprotein-1 (LAMP-1) 7 gctagcgccg ccacc atg gga atg cag gtg cag atccag agc ctg ttt ctg 51 Met Gly Met Gln Val Gln Ile Gln Ser Leu Phe Leu 15 10 ctc ctc ctg tgg gtg ccc ggg tcc aga gga atc agc cag gct gtg cac 99Leu Leu Leu Trp Val Pro Gly Ser Arg Gly Ile Ser Gln Ala Val His 15 20 25gcc gct cac gcc gaa atc aac gaa gct gga aga acc cct cca gct tat 147 AlaAla His Ala Glu Ile Asn Glu Ala Gly Arg Thr Pro Pro Ala Tyr 30 35 40 cgccct cca aac gct cct atc ctg ttc ttt ctg ctg acc aga atc ctg 195 Arg ProPro Asn Ala Pro Ile Leu Phe Phe Leu Leu Thr Arg Ile Leu 45 50 55 60 acaatc ccc cag tcc ctg gac gcc aag ttc gtg gct gcc tgg acc ctg 243 Thr IlePro Gln Ser Leu Asp Ala Lys Phe Val Ala Ala Trp Thr Leu 65 70 75 aag gctgcc gct aac aac atg ttg atc ccc att gct gtg ggc ggt gcc 291 Lys Ala AlaAla Asn Asn Met Leu Ile Pro Ile Ala Val Gly Gly Ala 80 85 90 ctg gca gggctg gtc ctc atc gtc ctc att gcc tac ctc att ggc agg 339 Leu Ala Gly LeuVal Leu Ile Val Leu Ile Ala Tyr Leu Ile Gly Arg 95 100 105 aag agg agtcac gcc ggc tat cag acc atc tagggtacc 378 Lys Arg Ser His Ala Gly TyrGln Thr Ile 110 115 8 118 PRT Artificial Sequence KappaLAMP-Th 8 Met GlyMet Gln Val Gln Ile Gln Ser Leu Phe Leu Leu Leu Leu Trp 1 5 10 15 ValPro Gly Ser Arg Gly Ile Ser Gln Ala Val His Ala Ala His Ala 20 25 30 GluIle Asn Glu Ala Gly Arg Thr Pro Pro Ala Tyr Arg Pro Pro Asn 35 40 45 AlaPro Ile Leu Phe Phe Leu Leu Thr Arg Ile Leu Thr Ile Pro Gln 50 55 60 SerLeu Asp Ala Lys Phe Val Ala Ala Trp Thr Leu Lys Ala Ala Ala 65 70 75 80Asn Asn Met Leu Ile Pro Ile Ala Val Gly Gly Ala Leu Ala Gly Leu 85 90 95Val Leu Ile Val Leu Ile Ala Tyr Leu Ile Gly Arg Lys Arg Ser His 100 105110 Ala Gly Tyr Gln Thr Ile 115 9 381 DNA Artificial Sequence H2M-Thconstruct encoding fusion of signal sequence of H2-M fused to multipleMHC class II epitopes and the cytoplasmic and transmembrane domains ofH2-M 9 gctagcgccg ccacc atg gct gca ctc tgg ctg ctg ctg ctg gtc ctc agt51 Met Ala Ala Leu Trp Leu Leu Leu Leu Val Leu Ser 1 5 10 ctg cac tgtatg ggg atc agc cag gct gtg cac gcc gct cac gcc gaa 99 Leu His Cys MetGly Ile Ser Gln Ala Val His Ala Ala His Ala Glu 15 20 25 atc aac gaa gctgga aga acc cct cca gct tat cgc cct cca aac gct 147 Ile Asn Glu Ala GlyArg Thr Pro Pro Ala Tyr Arg Pro Pro Asn Ala 30 35 40 cct atc ctg ttc tttctg ctg acc aga atc ctg aca atc ccc cag tcc 195 Pro Ile Leu Phe Phe LeuLeu Thr Arg Ile Leu Thr Ile Pro Gln Ser 45 50 55 60 ctg gac gcc aag ttcgtg gct gcc tgg acc ctg aag gct gcc gct aag 243 Leu Asp Ala Lys Phe ValAla Ala Trp Thr Leu Lys Ala Ala Ala Lys 65 70 75 gtc tct gtg tct gca gccacc ctg ggc ctg ggc ttc atc atc ttc tgt 291 Val Ser Val Ser Ala Ala ThrLeu Gly Leu Gly Phe Ile Ile Phe Cys 80 85 90 gtt ggc ttc ttc aga tgg cgcaag tct cat tcc tcc agc tac act cct 339 Val Gly Phe Phe Arg Trp Arg LysSer His Ser Ser Ser Tyr Thr Pro 95 100 105 ctc cct gga tcc acc tac ccagaa gga cgg cat tagggtacc 381 Leu Pro Gly Ser Thr Tyr Pro Glu Gly ArgHis 110 115 10 119 PRT Artificial Sequence H2M-Th 10 Met Ala Ala Leu TrpLeu Leu Leu Leu Val Leu Ser Leu His Cys Met 1 5 10 15 Gly Ile Ser GlnAla Val His Ala Ala His Ala Glu Ile Asn Glu Ala 20 25 30 Gly Arg Thr ProPro Ala Tyr Arg Pro Pro Asn Ala Pro Ile Leu Phe 35 40 45 Phe Leu Leu ThrArg Ile Leu Thr Ile Pro Gln Ser Leu Asp Ala Lys 50 55 60 Phe Val Ala AlaTrp Thr Leu Lys Ala Ala Ala Lys Val Ser Val Ser 65 70 75 80 Ala Ala ThrLeu Gly Leu Gly Phe Ile Ile Phe Cys Val Gly Phe Phe 85 90 95 Arg Trp ArgLys Ser His Ser Ser Ser Tyr Thr Pro Leu Pro Gly Ser 100 105 110 Thr TyrPro Glu Gly Arg His 115 11 432 DNA Artificial Sequence H2O-Th constructencoding fusion of signal sequence of H2-DO fused to multiple MHC classII epitopes and the cytoplasmic and transmembrane domains of H2-DO 11gctagcgccg ccacc atg ggc gct ggg agg gcc ccc tgg gtg gtg gct ctg 51 MetGly Ala Gly Arg Ala Pro Trp Val Val Ala Leu 1 5 10 ttg gtg aac ctc atgagg ctg gat tcc atc agc cag gct gtg cac gcc 99 Leu Val Asn Leu Met ArgLeu Asp Ser Ile Ser Gln Ala Val His Ala 15 20 25 gct cac gcc gaa atc aacgaa gct gga aga acc cct cca gct tat cgc 147 Ala His Ala Glu Ile Asn GluAla Gly Arg Thr Pro Pro Ala Tyr Arg 30 35 40 cct cca aac gct cct atc ctgttc ttt ctg ctg acc aga atc ctg aca 195 Pro Pro Asn Ala Pro Ile Leu PhePhe Leu Leu Thr Arg Ile Leu Thr 45 50 55 60 atc ccc cag tcc ctg gac gccaag ttc gtg gct gcc tgg acc ctg aag 243 Ile Pro Gln Ser Leu Asp Ala LysPhe Val Ala Ala Trp Thr Leu Lys 65 70 75 gct gcc gct ata ctg agt gga gctgca gtg ttc ctg ctt ggg ctg att 291 Ala Ala Ala Ile Leu Ser Gly Ala AlaVal Phe Leu Leu Gly Leu Ile 80 85 90 gtc ttc ctg gtg ggg gtt gtt atc catctc aag gct cag aaa gca tct 339 Val Phe Leu Val Gly Val Val Ile His LeuLys Ala Gln Lys Ala Ser 95 100 105 gtg gag act cag cct ggc aat gag agtagg tcc cgg atg atg gag cgg 387 Val Glu Thr Gln Pro Gly Asn Glu Ser ArgSer Arg Met Met Glu Arg 110 115 120 cta acc aag ttc aag gct gga ccg ggacat gtc aca tgaggtacc 432 Leu Thr Lys Phe Lys Ala Gly Pro Gly His ValThr 125 130 135 12 136 PRT Artificial Sequence H2O-Th 12 Met Gly Ala GlyArg Ala Pro Trp Val Val Ala Leu Leu Val Asn Leu 1 5 10 15 Met Arg LeuAsp Ser Ile Ser Gln Ala Val His Ala Ala His Ala Glu 20 25 30 Ile Asn GluAla Gly Arg Thr Pro Pro Ala Tyr Arg Pro Pro Asn Ala 35 40 45 Pro Ile LeuPhe Phe Leu Leu Thr Arg Ile Leu Thr Ile Pro Gln Ser 50 55 60 Leu Asp AlaLys Phe Val Ala Ala Trp Thr Leu Lys Ala Ala Ala Ile 65 70 75 80 Leu SerGly Ala Ala Val Phe Leu Leu Gly Leu Ile Val Phe Leu Val 85 90 95 Gly ValVal Ile His Leu Lys Ala Gln Lys Ala Ser Val Glu Thr Gln 100 105 110 ProGly Asn Glu Ser Arg Ser Arg Met Met Glu Arg Leu Thr Lys Phe 115 120 125Lys Ala Gly Pro Gly His Val Thr 130 135 13 816 DNA Artificial SequencePADRE-Influenza matrix construct encoding fusion of pan DR epitopesequence to amino-terminus of influenza matrix protein gene 13gctagcgccg ccacc atg gcc aag ttc gtg gct gcc tgg acc ctg aag gct 51 MetAla Lys Phe Val Ala Ala Trp Thr Leu Lys Ala 1 5 10 gcc gct atg agt cttcta acc gag gtc gaa acg tac gtt ctc tct atc 99 Ala Ala Met Ser Leu LeuThr Glu Val Glu Thr Tyr Val Leu Ser Ile 15 20 25 atc cca tca ggc ccc ctcaaa gcc gag atc gcg cag aga ctt gag gat 147 Ile Pro Ser Gly Pro Leu LysAla Glu Ile Ala Gln Arg Leu Glu Asp 30 35 40 gtt ttt gca ggg aag aac acagat ctt gag gct ctc atg gaa tgg cta 195 Val Phe Ala Gly Lys Asn Thr AspLeu Glu Ala Leu Met Glu Trp Leu 45 50 55 60 aag aca aga cca atc ctg tcacct ctg act aag gga att tta ggg ttt 243 Lys Thr Arg Pro Ile Leu Ser ProLeu Thr Lys Gly Ile Leu Gly Phe 65 70 75 gtg ttc acg ctc acc gtg ccc agtgag cga gga ctg cag cgt aga cga 291 Val Phe Thr Leu Thr Val Pro Ser GluArg Gly Leu Gln Arg Arg Arg 80 85 90 ttt gtc caa aat gcc cta aat ggg aatgga gac cca aac aac atg gac 339 Phe Val Gln Asn Ala Leu Asn Gly Asn GlyAsp Pro Asn Asn Met Asp 95 100 105 agg gca gtt aaa cta tac aag aag ctgaag agg gaa atg aca ttc cat 387 Arg Ala Val Lys Leu Tyr Lys Lys Leu LysArg Glu Met Thr Phe His 110 115 120 gga gca aag gaa gtt gca ctc agt tactca act ggt gcg ctt gcc agt 435 Gly Ala Lys Glu Val Ala Leu Ser Tyr SerThr Gly Ala Leu Ala Ser 125 130 135 140 tgc atg ggt ctc ata tac aac cggatg gga aca gtg acc aca gaa gtg 483 Cys Met Gly Leu Ile Tyr Asn Arg MetGly Thr Val Thr Thr Glu Val 145 150 155 gct ctt ggc cta gta tgt gcc acttgt gag cag att gct gat gcc caa 531 Ala Leu Gly Leu Val Cys Ala Thr CysGlu Gln Ile Ala Asp Ala Gln 160 165 170 cat cgg tcc cac agg cag atg gcgact acc acc aac cca cta atc agg 579 His Arg Ser His Arg Gln Met Ala ThrThr Thr Asn Pro Leu Ile Arg 175 180 185 cat gag aac aga atg gta cta gccagc act acg gct aag gcc atg gag 627 His Glu Asn Arg Met Val Leu Ala SerThr Thr Ala Lys Ala Met Glu 190 195 200 caa atg gct gga tca agt gag caggca gca gag gcc atg gaa gtc gca 675 Gln Met Ala Gly Ser Ser Glu Gln AlaAla Glu Ala Met Glu Val Ala 205 210 215 220 agt cag gct aga caa atg gtgcag gca atg agg aca att ggg act cac 723 Ser Gln Ala Arg Gln Met Val GlnAla Met Arg Thr Ile Gly Thr His 225 230 235 cct agc tcc agt gca ggt ctaaaa gat gat ctt att gaa aat ttg cag 771 Pro Ser Ser Ser Ala Gly Leu LysAsp Asp Leu Ile Glu Asn Leu Gln 240 245 250 gct tac cag aaa cgg atg ggggtg cag atg cag cga ttc aag 813 Ala Tyr Gln Lys Arg Met Gly Val Gln MetGln Arg Phe Lys 255 260 265 tga 816 14 266 PRT Artificial SequencePADRE-Influenza matrix 14 Met Ala Lys Phe Val Ala Ala Trp Thr Leu LysAla Ala Ala Met Ser 1 5 10 15 Leu Leu Thr Glu Val Glu Thr Tyr Val LeuSer Ile Ile Pro Ser Gly 20 25 30 Pro Leu Lys Ala Glu Ile Ala Gln Arg LeuGlu Asp Val Phe Ala Gly 35 40 45 Lys Asn Thr Asp Leu Glu Ala Leu Met GluTrp Leu Lys Thr Arg Pro 50 55 60 Ile Leu Ser Pro Leu Thr Lys Gly Ile LeuGly Phe Val Phe Thr Leu 65 70 75 80 Thr Val Pro Ser Glu Arg Gly Leu GlnArg Arg Arg Phe Val Gln Asn 85 90 95 Ala Leu Asn Gly Asn Gly Asp Pro AsnAsn Met Asp Arg Ala Val Lys 100 105 110 Leu Tyr Lys Lys Leu Lys Arg GluMet Thr Phe His Gly Ala Lys Glu 115 120 125 Val Ala Leu Ser Tyr Ser ThrGly Ala Leu Ala Ser Cys Met Gly Leu 130 135 140 Ile Tyr Asn Arg Met GlyThr Val Thr Thr Glu Val Ala Leu Gly Leu 145 150 155 160 Val Cys Ala ThrCys Glu Gln Ile Ala Asp Ala Gln His Arg Ser His 165 170 175 Arg Gln MetAla Thr Thr Thr Asn Pro Leu Ile Arg His Glu Asn Arg 180 185 190 Met ValLeu Ala Ser Thr Thr Ala Lys Ala Met Glu Gln Met Ala Gly 195 200 205 SerSer Glu Gln Ala Ala Glu Ala Met Glu Val Ala Ser Gln Ala Arg 210 215 220Gln Met Val Gln Ala Met Arg Thr Ile Gly Thr His Pro Ser Ser Ser 225 230235 240 Ala Gly Leu Lys Asp Asp Leu Ile Glu Asn Leu Gln Ala Tyr Gln Lys245 250 255 Arg Met Gly Val Gln Met Gln Arg Phe Lys 260 265 15 801 DNAArtificial Sequence PADRE-HBV-s construct encoding fusion of pan DRepitope sequence to amino-terminus of hepatitis B virus surface antigengene 15 gctagcgccg ccacc atg gcc aag ttc gtg gct gcc tgg acc ctg aag gct51 Met Ala Lys Phe Val Ala Ala Trp Thr Leu Lys Ala 1 5 10 gcc gct ctcgag att ggg gga ccc tgc ctg aac gcc gag aac atc aca 99 Ala Ala Leu GluIle Gly Gly Pro Cys Leu Asn Ala Glu Asn Ile Thr 15 20 25 tca gga ttc ctagga ccc ctt ctc gtg tta cag gcg ggg ttt ttc ttg 147 Ser Gly Phe Leu GlyPro Leu Leu Val Leu Gln Ala Gly Phe Phe Leu 30 35 40 ttg aca aga atc ctcaca ata ccg cag agt cta gac tcg tgg tgg act 195 Leu Thr Arg Ile Leu ThrIle Pro Gln Ser Leu Asp Ser Trp Trp Thr 45 50 55 60 tct ctc aat ttt ctaggg gga act acc gtg tgt ctt ggc caa aat tcg 243 Ser Leu Asn Phe Leu GlyGly Thr Thr Val Cys Leu Gly Gln Asn Ser 65 70 75 cag tcc cca acc tcc aatcac tca cca acc tct tgt cct cca act tgt 291 Gln Ser Pro Thr Ser Asn HisSer Pro Thr Ser Cys Pro Pro Thr Cys 80 85 90 cct ggt tat cgc tgg atg tgtctg cgg cgt ttt atc atc ttc ctc ttc 339 Pro Gly Tyr Arg Trp Met Cys LeuArg Arg Phe Ile Ile Phe Leu Phe 95 100 105 atc ctg ctg cta tgc ctc atcttc ttg ttg gtt ctt ctg gac tat caa 387 Ile Leu Leu Leu Cys Leu Ile PheLeu Leu Val Leu Leu Asp Tyr Gln 110 115 120 ggt atg ttg ccc gtt tgt cctcta att cca gga tcc tca aca acc agc 435 Gly Met Leu Pro Val Cys Pro LeuIle Pro Gly Ser Ser Thr Thr Ser 125 130 135 140 acg gga cca tgc cgg acctgc atg act act gct caa gga acc tct atg 483 Thr Gly Pro Cys Arg Thr CysMet Thr Thr Ala Gln Gly Thr Ser Met 145 150 155 tat ccc tcc tgt tgc tgtacc aaa cct tcg gac gga aat tgc acc tgt 531 Tyr Pro Ser Cys Cys Cys ThrLys Pro Ser Asp Gly Asn Cys Thr Cys 160 165 170 att ccc atc cca tca tcctgg gct ttc gga aaa ttc cta tgg gag tgg 579 Ile Pro Ile Pro Ser Ser TrpAla Phe Gly Lys Phe Leu Trp Glu Trp 175 180 185 gcc tca gcc cgt ttc tcctgg ctc agt tta cta gtg cca ttt gtt cag 627 Ala Ser Ala Arg Phe Ser TrpLeu Ser Leu Leu Val Pro Phe Val Gln 190 195 200 tgg ttc gta ggg ctt tccccc act gtt tgg ctt tca gtt ata tgg atg 675 Trp Phe Val Gly Leu Ser ProThr Val Trp Leu Ser Val Ile Trp Met 205 210 215 220 atg tgg tat tgg gggcca agt ctg tac agc atc ttg agt ccc ttt tta 723 Met Trp Tyr Trp Gly ProSer Leu Tyr Ser Ile Leu Ser Pro Phe Leu 225 230 235 ccg ctg tta cca attttc ttt tgt ctt tgg gta tac att taaaccctaa 772 Pro Leu Leu Pro Ile PhePhe Cys Leu Trp Val Tyr Ile 240 245 caaaacaaag agatggggtt actctctaa 80116 249 PRT Artificial Sequence PADRE-HBV-s 16 Met Ala Lys Phe Val AlaAla Trp Thr Leu Lys Ala Ala Ala Leu Glu 1 5 10 15 Ile Gly Gly Pro CysLeu Asn Ala Glu Asn Ile Thr Ser Gly Phe Leu 20 25 30 Gly Pro Leu Leu ValLeu Gln Ala Gly Phe Phe Leu Leu Thr Arg Ile 35 40 45 Leu Thr Ile Pro GlnSer Leu Asp Ser Trp Trp Thr Ser Leu Asn Phe 50 55 60 Leu Gly Gly Thr ThrVal Cys Leu Gly Gln Asn Ser Gln Ser Pro Thr 65 70 75 80 Ser Asn His SerPro Thr Ser Cys Pro Pro Thr Cys Pro Gly Tyr Arg 85 90 95 Trp Met Cys LeuArg Arg Phe Ile Ile Phe Leu Phe Ile Leu Leu Leu 100 105 110 Cys Leu IlePhe Leu Leu Val Leu Leu Asp Tyr Gln Gly Met Leu Pro 115 120 125 Val CysPro Leu Ile Pro Gly Ser Ser Thr Thr Ser Thr Gly Pro Cys 130 135 140 ArgThr Cys Met Thr Thr Ala Gln Gly Thr Ser Met Tyr Pro Ser Cys 145 150 155160 Cys Cys Thr Lys Pro Ser Asp Gly Asn Cys Thr Cys Ile Pro Ile Pro 165170 175 Ser Ser Trp Ala Phe Gly Lys Phe Leu Trp Glu Trp Ala Ser Ala Arg180 185 190 Phe Ser Trp Leu Ser Leu Leu Val Pro Phe Val Gln Trp Phe ValGly 195 200 205 Leu Ser Pro Thr Val Trp Leu Ser Val Ile Trp Met Met TrpTyr Trp 210 215 220 Gly Pro Ser Leu Tyr Ser Ile Leu Ser Pro Phe Leu ProLeu Leu Pro 225 230 235 240 Ile Phe Phe Cys Leu Trp Val Tyr Ile 245 17516 DNA Artificial Sequence Ig-alphaTh construct encoding fusion of thesignal sequence of Ig-alpha protein fused to multiple MHC class IIepitopes and the transmembrane and cytoplasmic domains of the Ig-alphaprotein 17 gctagcgccg ccacc atg cca ggg ggt cta gaa gcc ctc aga gcc ctgcct 51 Met Pro Gly Gly Leu Glu Ala Leu Arg Ala Leu Pro 1 5 10 ctc ctcctc ttc ttg tca tac gcc tgt ttg ggt ccc gga tgc cag gcc 99 Leu Leu LeuPhe Leu Ser Tyr Ala Cys Leu Gly Pro Gly Cys Gln Ala 15 20 25 atc agc caggct gtg cac gcc gct cac gcc gaa atc aac gaa gct gga 147 Ile Ser Gln AlaVal His Ala Ala His Ala Glu Ile Asn Glu Ala Gly 30 35 40 aga acc cct ccagct tat cgc cct cca aac gct cct atc ctg ttc ttt 195 Arg Thr Pro Pro AlaTyr Arg Pro Pro Asn Ala Pro Ile Leu Phe Phe 45 50 55 60 ctg ctg acc agaatc ctg aca atc ccc cag tcc ctg gac gcc aag ttc 243 Leu Leu Thr Arg IleLeu Thr Ile Pro Gln Ser Leu Asp Ala Lys Phe 65 70 75 gtg gct gcc tgg accctg aag gct gcc gct ggg atc atc ttg ctg ttc 291 Val Ala Ala Trp Thr LeuLys Ala Ala Ala Gly Ile Ile Leu Leu Phe 80 85 90 tgt gca gtg gtg cca gggacg ctg ctg cta ttc agg aaa cgg tgg caa 339 Cys Ala Val Val Pro Gly ThrLeu Leu Leu Phe Arg Lys Arg Trp Gln 95 100 105 aat gag aag ttt ggg gtggac atg cca gat gac tat gaa gat gaa aat 387 Asn Glu Lys Phe Gly Val AspMet Pro Asp Asp Tyr Glu Asp Glu Asn 110 115 120 ctc tat gag ggc ctg aacctt gat gac tgt tct atg tat gag gac atc 435 Leu Tyr Glu Gly Leu Asn LeuAsp Asp Cys Ser Met Tyr Glu Asp Ile 125 130 135 140 tcc agg gga ctc cagggc acc tac cag gat gtg ggc aac ctc cac att 483 Ser Arg Gly Leu Gln GlyThr Tyr Gln Asp Val Gly Asn Leu His Ile 145 150 155 gga gat gcc cag ctggaa aag cca tgaggtacc 516 Gly Asp Ala Gln Leu Glu Lys Pro 160 18 164 PRTArtificial Sequence Ig-alphaTh 18 Met Pro Gly Gly Leu Glu Ala Leu ArgAla Leu Pro Leu Leu Leu Phe 1 5 10 15 Leu Ser Tyr Ala Cys Leu Gly ProGly Cys Gln Ala Ile Ser Gln Ala 20 25 30 Val His Ala Ala His Ala Glu IleAsn Glu Ala Gly Arg Thr Pro Pro 35 40 45 Ala Tyr Arg Pro Pro Asn Ala ProIle Leu Phe Phe Leu Leu Thr Arg 50 55 60 Ile Leu Thr Ile Pro Gln Ser LeuAsp Ala Lys Phe Val Ala Ala Trp 65 70 75 80 Thr Leu Lys Ala Ala Ala GlyIle Ile Leu Leu Phe Cys Ala Val Val 85 90 95 Pro Gly Thr Leu Leu Leu PheArg Lys Arg Trp Gln Asn Glu Lys Phe 100 105 110 Gly Val Asp Met Pro AspAsp Tyr Glu Asp Glu Asn Leu Tyr Glu Gly 115 120 125 Leu Asn Leu Asp AspCys Ser Met Tyr Glu Asp Ile Ser Arg Gly Leu 130 135 140 Gln Gly Thr TyrGln Asp Val Gly Asn Leu His Ile Gly Asp Ala Gln 145 150 155 160 Leu GluLys Pro 19 480 DNA Artificial Sequence Ig-betaTh construct encodingfusion of the signal sequence of Ig-beta protein fused to multiple MHCclass II epitopes and the transmembrane and cytoplasmic domains of theIg-beta protein 19 gctagcgccg ccacc atg gcc aca ctg gtg ctg tct tcc atgccc tgc cac 51 Met Ala Thr Leu Val Leu Ser Ser Met Pro Cys His 1 5 10tgg ctg ttg ttc ctg ctg ctg ctc ttc tca ggt gag ccg atc agc cag 99 TrpLeu Leu Phe Leu Leu Leu Leu Phe Ser Gly Glu Pro Ile Ser Gln 15 20 25 gctgtg cac gcc gct cac gcc gaa atc aac gaa gct gga aga acc cct 147 Ala ValHis Ala Ala His Ala Glu Ile Asn Glu Ala Gly Arg Thr Pro 30 35 40 cca gcttat cgc cct cca aac gct cct atc ctg ttc ttt ctg ctg acc 195 Pro Ala TyrArg Pro Pro Asn Ala Pro Ile Leu Phe Phe Leu Leu Thr 45 50 55 60 aga atcctg aca atc ccc cag tcc ctg gac gcc aag ttc gtg gct gcc 243 Arg Ile LeuThr Ile Pro Gln Ser Leu Asp Ala Lys Phe Val Ala Ala 65 70 75 tgg acc ctgaag gct gcc gct att atc ttg atc cag acc ctc ctc atc 291 Trp Thr Leu LysAla Ala Ala Ile Ile Leu Ile Gln Thr Leu Leu Ile 80 85 90 atc ctc ttc atcatt gtg ccc atc ttc ctg cta ctt gac aag gat gac 339 Ile Leu Phe Ile IleVal Pro Ile Phe Leu Leu Leu Asp Lys Asp Asp 95 100 105 ggc aag gct gggatg gag gaa gat cac acc tat gag ggc ttg aac att 387 Gly Lys Ala Gly MetGlu Glu Asp His Thr Tyr Glu Gly Leu Asn Ile 110 115 120 gac cag aca gccacc tat gaa gac ata gtg act ctt cgg aca ggg gag 435 Asp Gln Thr Ala ThrTyr Glu Asp Ile Val Thr Leu Arg Thr Gly Glu 125 130 135 140 gta aag tggtcg gta gga gag cat cca ggc cag gaa tgaggtacc 480 Val Lys Trp Ser ValGly Glu His Pro Gly Gln Glu 145 150 20 152 PRT Artificial SequenceIg-betaTh 20 Met Ala Thr Leu Val Leu Ser Ser Met Pro Cys His Trp Leu LeuPhe 1 5 10 15 Leu Leu Leu Leu Phe Ser Gly Glu Pro Ile Ser Gln Ala ValHis Ala 20 25 30 Ala His Ala Glu Ile Asn Glu Ala Gly Arg Thr Pro Pro AlaTyr Arg 35 40 45 Pro Pro Asn Ala Pro Ile Leu Phe Phe Leu Leu Thr Arg IleLeu Thr 50 55 60 Ile Pro Gln Ser Leu Asp Ala Lys Phe Val Ala Ala Trp ThrLeu Lys 65 70 75 80 Ala Ala Ala Ile Ile Leu Ile Gln Thr Leu Leu Ile IleLeu Phe Ile 85 90 95 Ile Val Pro Ile Phe Leu Leu Leu Asp Lys Asp Asp GlyLys Ala Gly 100 105 110 Met Glu Glu Asp His Thr Tyr Glu Gly Leu Asn IleAsp Gln Thr Ala 115 120 125 Thr Tyr Glu Asp Ile Val Thr Leu Arg Thr GlyGlu Val Lys Trp Ser 130 135 140 Val Gly Glu His Pro Gly Gln Glu 145 15021 264 DNA Artificial Sequence SigTh construct encoding fusion of thesignal sequence of kappa immunoglobulin fused to multiple MHC class IIepitopes 21 gctagcgccg ccacc atg gga atg cag gtg cag atc cag agc ctg tttctg 51 Met Gly Met Gln Val Gln Ile Gln Ser Leu Phe Leu 1 5 10 ctc ctcctg tgg gtg ccc ggg tcc cga gga atc agc cag gct gtg cac 99 Leu Leu LeuTrp Val Pro Gly Ser Arg Gly Ile Ser Gln Ala Val His 15 20 25 gcc gct cacgcc gaa atc aac gaa gct gga aga acc cct cca gct tat 147 Ala Ala His AlaGlu Ile Asn Glu Ala Gly Arg Thr Pro Pro Ala Tyr 30 35 40 cgc cct cca aacgct cct atc ctg ttc ttt ctg ctg acc aga atc ctg 195 Arg Pro Pro Asn AlaPro Ile Leu Phe Phe Leu Leu Thr Arg Ile Leu 45 50 55 60 aca atc ccc cagtcc ctg gac gcc aag ttc gtg gct gcc tgg acc ctg 243 Thr Ile Pro Gln SerLeu Asp Ala Lys Phe Val Ala Ala Trp Thr Leu 65 70 75 aag gct gcc gcttgaggtacc 264 Lys Ala Ala Ala 80 22 80 PRT Artificial Sequence SigTh 22Met Gly Met Gln Val Gln Ile Gln Ser Leu Phe Leu Leu Leu Leu Trp 1 5 1015 Val Pro Gly Ser Arg Gly Ile Ser Gln Ala Val His Ala Ala His Ala 20 2530 Glu Ile Asn Glu Ala Gly Arg Thr Pro Pro Ala Tyr Arg Pro Pro Asn 35 4045 Ala Pro Ile Leu Phe Phe Leu Leu Thr Arg Ile Leu Thr Ile Pro Gln 50 5560 Ser Leu Asp Ala Lys Phe Val Ala Ala Trp Thr Leu Lys Ala Ala Ala 65 7075 80 23 723 DNA Homo sapiens CDS (8)...(706) human HLA-DR invariantchain (Ii) protein 23 ttcccag atg cac agg agg aga agc agg agc tgt cgggaa gat cag aag 49 Met His Arg Arg Arg Ser Arg Ser Cys Arg Glu Asp GlnLys 1 5 10 cca gtc atg gat gac cag cgc gac ctt atc tcc aac aat gag caactg 97 Pro Val Met Asp Asp Gln Arg Asp Leu Ile Ser Asn Asn Glu Gln Leu15 20 25 30 ccc atg ctg ggc cgg cgc cct ggg gcc ccg gag agc aag tgc agccgc 145 Pro Met Leu Gly Arg Arg Pro Gly Ala Pro Glu Ser Lys Cys Ser Arg35 40 45 gga gcc ctg tac aca ggc ttt tcc atc ctg gtg act ctg ctc ctc gct193 Gly Ala Leu Tyr Thr Gly Phe Ser Ile Leu Val Thr Leu Leu Leu Ala 5055 60 ggc cag gcc acc acc gcc tac ttc ctg tac cag cag cag ggc cgg ctg241 Gly Gln Ala Thr Thr Ala Tyr Phe Leu Tyr Gln Gln Gln Gly Arg Leu 6570 75 gac aaa ctg aca gtc acc tcc cag aac ctg cag ctg gag aac ctg cgc289 Asp Lys Leu Thr Val Thr Ser Gln Asn Leu Gln Leu Glu Asn Leu Arg 8085 90 atg aag ctt ccc aag cct ccc aag cct gtg agc aag atg cgc atg gcc337 Met Lys Leu Pro Lys Pro Pro Lys Pro Val Ser Lys Met Arg Met Ala 95100 105 110 acc ccg ctg ctg atg cag gcg ctg ccc atg gga gcc ctg ccc cagggg 385 Thr Pro Leu Leu Met Gln Ala Leu Pro Met Gly Ala Leu Pro Gln Gly115 120 125 ccc atg cag aat gcc acc aag tat ggc aac atg aca gag gac catgtg 433 Pro Met Gln Asn Ala Thr Lys Tyr Gly Asn Met Thr Glu Asp His Val130 135 140 atg cac ctg ctc cag aat gct gac ccc ctg aag gtg tac ccg ccactg 481 Met His Leu Leu Gln Asn Ala Asp Pro Leu Lys Val Tyr Pro Pro Leu145 150 155 aag ggg agc ttc ccg gag aac ctg aga cac ctt aag aac acc atggag 529 Lys Gly Ser Phe Pro Glu Asn Leu Arg His Leu Lys Asn Thr Met Glu160 165 170 acc ata gac tgg aag gtc ttt gag agc tgg atg cac cat tgg ctcctg 577 Thr Ile Asp Trp Lys Val Phe Glu Ser Trp Met His His Trp Leu Leu175 180 185 190 ttt gaa atg agc agg cac tcc ttg gag caa aag ccc act gacgct cca 625 Phe Glu Met Ser Arg His Ser Leu Glu Gln Lys Pro Thr Asp AlaPro 195 200 205 ccg aaa gag tca ctg gaa ctg gag gac ccg tct tct ggg ctgggt gtg 673 Pro Lys Glu Ser Leu Glu Leu Glu Asp Pro Ser Ser Gly Leu GlyVal 210 215 220 acc aag cag gat ctg ggc cca gtc ccc atg tgagagcagcagaggcggtc 723 Thr Lys Gln Asp Leu Gly Pro Val Pro Met 225 230 24 232PRT Homo sapiens human HLA-DR invariant chain (Ii) protein 24 Met HisArg Arg Arg Ser Arg Ser Cys Arg Glu Asp Gln Lys Pro Val 1 5 10 15 MetAsp Asp Gln Arg Asp Leu Ile Ser Asn Asn Glu Gln Leu Pro Met 20 25 30 LeuGly Arg Arg Pro Gly Ala Pro Glu Ser Lys Cys Ser Arg Gly Ala 35 40 45 LeuTyr Thr Gly Phe Ser Ile Leu Val Thr Leu Leu Leu Ala Gly Gln 50 55 60 AlaThr Thr Ala Tyr Phe Leu Tyr Gln Gln Gln Gly Arg Leu Asp Lys 65 70 75 80Leu Thr Val Thr Ser Gln Asn Leu Gln Leu Glu Asn Leu Arg Met Lys 85 90 95Leu Pro Lys Pro Pro Lys Pro Val Ser Lys Met Arg Met Ala Thr Pro 100 105110 Leu Leu Met Gln Ala Leu Pro Met Gly Ala Leu Pro Gln Gly Pro Met 115120 125 Gln Asn Ala Thr Lys Tyr Gly Asn Met Thr Glu Asp His Val Met His130 135 140 Leu Leu Gln Asn Ala Asp Pro Leu Lys Val Tyr Pro Pro Leu LysGly 145 150 155 160 Ser Phe Pro Glu Asn Leu Arg His Leu Lys Asn Thr MetGlu Thr Ile 165 170 175 Asp Trp Lys Val Phe Glu Ser Trp Met His His TrpLeu Leu Phe Glu 180 185 190 Met Ser Arg His Ser Leu Glu Gln Lys Pro ThrAsp Ala Pro Pro Lys 195 200 205 Glu Ser Leu Glu Leu Glu Asp Pro Ser SerGly Leu Gly Val Thr Lys 210 215 220 Gln Asp Leu Gly Pro Val Pro Met 225230 25 1298 DNA Homo sapiens CDS (11)...(1261) human lysosomal membraneglycoprotein-1 (LAMP-1) 25 ccgcctcggc atg gcg ccc cgc agc gcc cgg cgaccc ctg ctg ctg cta 49 Met Ala Pro Arg Ser Ala Arg Arg Pro Leu Leu LeuLeu 1 5 10 ctg cct gtt gct gct gct cgg cct cat gca ttg tcg tca gca gccatg 97 Leu Pro Val Ala Ala Ala Arg Pro His Ala Leu Ser Ser Ala Ala Met15 20 25 ttt atg gtg aaa aat ggc aac ggg acc gcg tgc ata atg gcc aac ttc145 Phe Met Val Lys Asn Gly Asn Gly Thr Ala Cys Ile Met Ala Asn Phe 3035 40 45 tct gct gcc ttc tca gtg aac tac gac acc aag agt ggc ccc aag aac193 Ser Ala Ala Phe Ser Val Asn Tyr Asp Thr Lys Ser Gly Pro Lys Asn 5055 60 atg acc ttt gac ctg cca tca gat gcc aca gtg gtg ctc aac cgc agc241 Met Thr Phe Asp Leu Pro Ser Asp Ala Thr Val Val Leu Asn Arg Ser 6570 75 tcc tgt gga aaa gag aac act tct gac ccc agt ctc gtg att gct ttt289 Ser Cys Gly Lys Glu Asn Thr Ser Asp Pro Ser Leu Val Ile Ala Phe 8085 90 gga aga gga cat aca ctc act ctc aat ttc acg aga aat gca aca cgt337 Gly Arg Gly His Thr Leu Thr Leu Asn Phe Thr Arg Asn Ala Thr Arg 95100 105 tac agc gtt cag ctc atg agt ttt gtt tat aac ttg tca gac aca cac385 Tyr Ser Val Gln Leu Met Ser Phe Val Tyr Asn Leu Ser Asp Thr His 110115 120 125 ctt ttc ccc aat gcg agc tcc aaa gaa atc aag act gtg gaa tctata 433 Leu Phe Pro Asn Ala Ser Ser Lys Glu Ile Lys Thr Val Glu Ser Ile130 135 140 act gac atc agg gca gat ata gat aaa aaa tac aga tgt gtt agtggc 481 Thr Asp Ile Arg Ala Asp Ile Asp Lys Lys Tyr Arg Cys Val Ser Gly145 150 155 acc cag gtc cac atg aac aac gtg acc gta acg ctc cat gat gccacc 529 Thr Gln Val His Met Asn Asn Val Thr Val Thr Leu His Asp Ala Thr160 165 170 atc cag gcg tac ctt tcc aac agc agc ttc agc agg gga gag acacgc 577 Ile Gln Ala Tyr Leu Ser Asn Ser Ser Phe Ser Arg Gly Glu Thr Arg175 180 185 tgt gaa caa gac agg cct tcc cca acc aca gcg ccc cct gcg ccaccc 625 Cys Glu Gln Asp Arg Pro Ser Pro Thr Thr Ala Pro Pro Ala Pro Pro190 195 200 205 agc ccc tcg ccc tca ccc gtg ccc aag agc ccc tct gtg gacaag tac 673 Ser Pro Ser Pro Ser Pro Val Pro Lys Ser Pro Ser Val Asp LysTyr 210 215 220 aac gtg agc ggc acc aac ggg acc tgc ctg ctg gcc agc atgggg ctg 721 Asn Val Ser Gly Thr Asn Gly Thr Cys Leu Leu Ala Ser Met GlyLeu 225 230 235 cag ctg aac ctc acc tat gag agg aag gac aac acg acg gtgaca agg 769 Gln Leu Asn Leu Thr Tyr Glu Arg Lys Asp Asn Thr Thr Val ThrArg 240 245 250 ctt ctc aac atc aac ccc aac aag acc tcg gcc agc ggg agctgc ggc 817 Leu Leu Asn Ile Asn Pro Asn Lys Thr Ser Ala Ser Gly Ser CysGly 255 260 265 gcc cac ctg gtg act ctg gag ctg cac agc gag ggc acc accgtc ctg 865 Ala His Leu Val Thr Leu Glu Leu His Ser Glu Gly Thr Thr ValLeu 270 275 280 285 ctc ttc cag ttc ggg atg aat gca agt tct agc cgg tttttc cta caa 913 Leu Phe Gln Phe Gly Met Asn Ala Ser Ser Ser Arg Phe PheLeu Gln 290 295 300 gga atc cag ttg aat aca att ctt cct gac gcc aga gaccct gcc ttt 961 Gly Ile Gln Leu Asn Thr Ile Leu Pro Asp Ala Arg Asp ProAla Phe 305 310 315 aaa gct gcc aac ggc tcc ctg cga gcg ctg cag gcc acagtc ggc aat 1009 Lys Ala Ala Asn Gly Ser Leu Arg Ala Leu Gln Ala Thr ValGly Asn 320 325 330 tcc tac aag tgc aac gcg gag gag cac gtc cgt gtc acgaag gcg ttt 1057 Ser Tyr Lys Cys Asn Ala Glu Glu His Val Arg Val Thr LysAla Phe 335 340 345 tca gtc aat ata ttc aaa gtg tgg gtc cag gct ttc aaggtg gaa ggt 1105 Ser Val Asn Ile Phe Lys Val Trp Val Gln Ala Phe Lys ValGlu Gly 350 355 360 365 ggc cag ttt ggc tct gtg gag gag tgt ctg ctg gacgag aac agc acg 1153 Gly Gln Phe Gly Ser Val Glu Glu Cys Leu Leu Asp GluAsn Ser Thr 370 375 380 ctg atc ccc atc gct gtg ggt ggt gcc ctg gcg gggctg gtc ctc atc 1201 Leu Ile Pro Ile Ala Val Gly Gly Ala Leu Ala Gly LeuVal Leu Ile 385 390 395 gtc ctc atc gcc tac ctc gtc ggc agg aag agg agtcac gca ggc tac 1249 Val Leu Ile Ala Tyr Leu Val Gly Arg Lys Arg Ser HisAla Gly Tyr 400 405 410 cag act atc tagcctggtg cacgcaggca cagcagctgcaggggcctct 1298 Gln Thr Ile 415 26 416 PRT Homo sapiens human lysosomalmembrane glycoprotein-1 (LAMP-1) 26 Met Ala Pro Arg Ser Ala Arg Arg ProLeu Leu Leu Leu Leu Pro Val 1 5 10 15 Ala Ala Ala Arg Pro His Ala LeuSer Ser Ala Ala Met Phe Met Val 20 25 30 Lys Asn Gly Asn Gly Thr Ala CysIle Met Ala Asn Phe Ser Ala Ala 35 40 45 Phe Ser Val Asn Tyr Asp Thr LysSer Gly Pro Lys Asn Met Thr Phe 50 55 60 Asp Leu Pro Ser Asp Ala Thr ValVal Leu Asn Arg Ser Ser Cys Gly 65 70 75 80 Lys Glu Asn Thr Ser Asp ProSer Leu Val Ile Ala Phe Gly Arg Gly 85 90 95 His Thr Leu Thr Leu Asn PheThr Arg Asn Ala Thr Arg Tyr Ser Val 100 105 110 Gln Leu Met Ser Phe ValTyr Asn Leu Ser Asp Thr His Leu Phe Pro 115 120 125 Asn Ala Ser Ser LysGlu Ile Lys Thr Val Glu Ser Ile Thr Asp Ile 130 135 140 Arg Ala Asp IleAsp Lys Lys Tyr Arg Cys Val Ser Gly Thr Gln Val 145 150 155 160 His MetAsn Asn Val Thr Val Thr Leu His Asp Ala Thr Ile Gln Ala 165 170 175 TyrLeu Ser Asn Ser Ser Phe Ser Arg Gly Glu Thr Arg Cys Glu Gln 180 185 190Asp Arg Pro Ser Pro Thr Thr Ala Pro Pro Ala Pro Pro Ser Pro Ser 195 200205 Pro Ser Pro Val Pro Lys Ser Pro Ser Val Asp Lys Tyr Asn Val Ser 210215 220 Gly Thr Asn Gly Thr Cys Leu Leu Ala Ser Met Gly Leu Gln Leu Asn225 230 235 240 Leu Thr Tyr Glu Arg Lys Asp Asn Thr Thr Val Thr Arg LeuLeu Asn 245 250 255 Ile Asn Pro Asn Lys Thr Ser Ala Ser Gly Ser Cys GlyAla His Leu 260 265 270 Val Thr Leu Glu Leu His Ser Glu Gly Thr Thr ValLeu Leu Phe Gln 275 280 285 Phe Gly Met Asn Ala Ser Ser Ser Arg Phe PheLeu Gln Gly Ile Gln 290 295 300 Leu Asn Thr Ile Leu Pro Asp Ala Arg AspPro Ala Phe Lys Ala Ala 305 310 315 320 Asn Gly Ser Leu Arg Ala Leu GlnAla Thr Val Gly Asn Ser Tyr Lys 325 330 335 Cys Asn Ala Glu Glu His ValArg Val Thr Lys Ala Phe Ser Val Asn 340 345 350 Ile Phe Lys Val Trp ValGln Ala Phe Lys Val Glu Gly Gly Gln Phe 355 360 365 Gly Ser Val Glu GluCys Leu Leu Asp Glu Asn Ser Thr Leu Ile Pro 370 375 380 Ile Ala Val GlyGly Ala Leu Ala Gly Leu Val Leu Ile Val Leu Ile 385 390 395 400 Ala TyrLeu Val Gly Arg Lys Arg Ser His Ala Gly Tyr Gln Thr Ile 405 410 415 27792 DNA Homo sapiens CDS (1)...(792) human HLA-DMB 27 atg atc aca ttcctg ccg ctg ctg ctg ggg ctc agc ctg ggc tgc aca 48 Met Ile Thr Phe LeuPro Leu Leu Leu Gly Leu Ser Leu Gly Cys Thr 1 5 10 15 gga gca ggt ggcttc gtg gcc cat gtg gaa agc acc tgt ctg ttg gat 96 Gly Ala Gly Gly PheVal Ala His Val Glu Ser Thr Cys Leu Leu Asp 20 25 30 gat gct ggg act ccaaag gat ttc aca tac tgc atc tcc ttc aac aag 144 Asp Ala Gly Thr Pro LysAsp Phe Thr Tyr Cys Ile Ser Phe Asn Lys 35 40 45 gat ctg ctg acc tgc tgggat cca gag gag aat aag atg gcc cct tgc 192 Asp Leu Leu Thr Cys Trp AspPro Glu Glu Asn Lys Met Ala Pro Cys 50 55 60 gaa ttt ggg gtg ctg aat agcttg gcg aat gtc ctc tca cag cac ctc 240 Glu Phe Gly Val Leu Asn Ser LeuAla Asn Val Leu Ser Gln His Leu 65 70 75 80 aac caa aaa gac acc ctg atgcag cgc ttg cgc aat ggg ctt cag aat 288 Asn Gln Lys Asp Thr Leu Met GlnArg Leu Arg Asn Gly Leu Gln Asn 85 90 95 tgt gcc aca cac acc cag ccc ttctgg gga tca ctg acc aac agg aca 336 Cys Ala Thr His Thr Gln Pro Phe TrpGly Ser Leu Thr Asn Arg Thr 100 105 110 cgg cca cca tct gtg caa gta gccaaa acc act cct ttt aac acg agg 384 Arg Pro Pro Ser Val Gln Val Ala LysThr Thr Pro Phe Asn Thr Arg 115 120 125 gag cct gtg atg ctg gcc tgc tatgtg tgg ggc ttc tat cca gca gaa 432 Glu Pro Val Met Leu Ala Cys Tyr ValTrp Gly Phe Tyr Pro Ala Glu 130 135 140 gtg act atc acg tgg agg aag aacggg aag ctt gtc atg cct cac agc 480 Val Thr Ile Thr Trp Arg Lys Asn GlyLys Leu Val Met Pro His Ser 145 150 155 160 agt gcg cac aag act gcc cagccc aat gga gac tgg aca tac cag acc 528 Ser Ala His Lys Thr Ala Gln ProAsn Gly Asp Trp Thr Tyr Gln Thr 165 170 175 ctc tcc cat tta gcc tta accccc tct tac ggg gac act tac acc tgt 576 Leu Ser His Leu Ala Leu Thr ProSer Tyr Gly Asp Thr Tyr Thr Cys 180 185 190 gtg gta gag cac att ggg gctcct gag ccc atc ctt cgg gac tgg aca 624 Val Val Glu His Ile Gly Ala ProGlu Pro Ile Leu Arg Asp Trp Thr 195 200 205 cct ggg ctg tcc ccc atg cagacc ctg aag gtt tct gtg tct gca gtg 672 Pro Gly Leu Ser Pro Met Gln ThrLeu Lys Val Ser Val Ser Ala Val 210 215 220 act ctg ggc ctg ggc ctc atcatc ttc tct ctt ggt gtg atc agc tgg 720 Thr Leu Gly Leu Gly Leu Ile IlePhe Ser Leu Gly Val Ile Ser Trp 225 230 235 240 cgg aga gct ggc cac tctagt tac act cct ctt cct ggg tcc aat tat 768 Arg Arg Ala Gly His Ser SerTyr Thr Pro Leu Pro Gly Ser Asn Tyr 245 250 255 tca gaa gga tgg cac atttcc tag 792 Ser Glu Gly Trp His Ile Ser 260 28 263 PRT Homo sapienshuman HLA-DMB 28 Met Ile Thr Phe Leu Pro Leu Leu Leu Gly Leu Ser Leu GlyCys Thr 1 5 10 15 Gly Ala Gly Gly Phe Val Ala His Val Glu Ser Thr CysLeu Leu Asp 20 25 30 Asp Ala Gly Thr Pro Lys Asp Phe Thr Tyr Cys Ile SerPhe Asn Lys 35 40 45 Asp Leu Leu Thr Cys Trp Asp Pro Glu Glu Asn Lys MetAla Pro Cys 50 55 60 Glu Phe Gly Val Leu Asn Ser Leu Ala Asn Val Leu SerGln His Leu 65 70 75 80 Asn Gln Lys Asp Thr Leu Met Gln Arg Leu Arg AsnGly Leu Gln Asn 85 90 95 Cys Ala Thr His Thr Gln Pro Phe Trp Gly Ser LeuThr Asn Arg Thr 100 105 110 Arg Pro Pro Ser Val Gln Val Ala Lys Thr ThrPro Phe Asn Thr Arg 115 120 125 Glu Pro Val Met Leu Ala Cys Tyr Val TrpGly Phe Tyr Pro Ala Glu 130 135 140 Val Thr Ile Thr Trp Arg Lys Asn GlyLys Leu Val Met Pro His Ser 145 150 155 160 Ser Ala His Lys Thr Ala GlnPro Asn Gly Asp Trp Thr Tyr Gln Thr 165 170 175 Leu Ser His Leu Ala LeuThr Pro Ser Tyr Gly Asp Thr Tyr Thr Cys 180 185 190 Val Val Glu His IleGly Ala Pro Glu Pro Ile Leu Arg Asp Trp Thr 195 200 205 Pro Gly Leu SerPro Met Gln Thr Leu Lys Val Ser Val Ser Ala Val 210 215 220 Thr Leu GlyLeu Gly Leu Ile Ile Phe Ser Leu Gly Val Ile Ser Trp 225 230 235 240 ArgArg Ala Gly His Ser Ser Tyr Thr Pro Leu Pro Gly Ser Asn Tyr 245 250 255Ser Glu Gly Trp His Ile Ser 260 29 822 DNA Homo sapiens CDS (1)...(822)human HLA-DO beta 29 atg ggt tct ggg tgg gtc ccc tgg gtg gtg gct ctg ctagtg aat ctg 48 Met Gly Ser Gly Trp Val Pro Trp Val Val Ala Leu Leu ValAsn Leu 1 5 10 15 acc caa ctg gat tcc tcc atg act caa ggc aca gac tctcca gaa gat 96 Thr Gln Leu Asp Ser Ser Met Thr Gln Gly Thr Asp Ser ProGlu Asp 20 25 30 ttt gtg att cag gca aag gct gac tgt tac ttc acc aac gggaca gaa 144 Phe Val Ile Gln Ala Lys Ala Asp Cys Tyr Phe Thr Asn Gly ThrGlu 35 40 45 aag gtg cag ttt gtg gtc aga ttc atc ttt aac ttg gag gag tatgta 192 Lys Val Gln Phe Val Val Arg Phe Ile Phe Asn Leu Glu Glu Tyr Val50 55 60 cgt ttc gac agt gat gtg ggg atg ttt gtg gca ttg acc aag ctg ggg240 Arg Phe Asp Ser Asp Val Gly Met Phe Val Ala Leu Thr Lys Leu Gly 6570 75 80 cag cca gat gct gag cag tgg aac agc cgg ctg gat ctc ttg gag agg288 Gln Pro Asp Ala Glu Gln Trp Asn Ser Arg Leu Asp Leu Leu Glu Arg 8590 95 agc aga cag gcc gtg gat ggg gtc tgt aga cac aac tac agg ctg ggc336 Ser Arg Gln Ala Val Asp Gly Val Cys Arg His Asn Tyr Arg Leu Gly 100105 110 gca ccc ttc act gtg ggg aga aaa gtg caa cca gag gtg aca gtg tac384 Ala Pro Phe Thr Val Gly Arg Lys Val Gln Pro Glu Val Thr Val Tyr 115120 125 cca gag agg acc cca ctc ctg cac cag cat aat ctg ctg cac tgc tct432 Pro Glu Arg Thr Pro Leu Leu His Gln His Asn Leu Leu His Cys Ser 130135 140 gtg aca ggc ttc tat cca ggg gat atc aag atc aag tgg ttc ctg aat480 Val Thr Gly Phe Tyr Pro Gly Asp Ile Lys Ile Lys Trp Phe Leu Asn 145150 155 160 ggg cag gag gag aga gct ggg gtc atg tcc act ggc cct atc aggaat 528 Gly Gln Glu Glu Arg Ala Gly Val Met Ser Thr Gly Pro Ile Arg Asn165 170 175 gga gac tgg acc ttt cag act gtg gtg atg cta gaa atg act cctgaa 576 Gly Asp Trp Thr Phe Gln Thr Val Val Met Leu Glu Met Thr Pro Glu180 185 190 ctt gga cat gtc tac acc tgc ctt gtc gat cac tcc agc ctg ctgagc 624 Leu Gly His Val Tyr Thr Cys Leu Val Asp His Ser Ser Leu Leu Ser195 200 205 cct gtt tct gtg gag tgg aga gct cag tct gaa tat tct tgg agaaag 672 Pro Val Ser Val Glu Trp Arg Ala Gln Ser Glu Tyr Ser Trp Arg Lys210 215 220 atg ctg agt ggc att gca gcc ttc cta ctt ggg cta atc ttc cttctg 720 Met Leu Ser Gly Ile Ala Ala Phe Leu Leu Gly Leu Ile Phe Leu Leu225 230 235 240 gtg gga atc gtc atc cag cta agg gct cag aaa gga tat gtgagg acg 768 Val Gly Ile Val Ile Gln Leu Arg Ala Gln Lys Gly Tyr Val ArgThr 245 250 255 cag atg tct ggt aat gag gtc tca aga gct gtt ctg ctc cctcag tca 816 Gln Met Ser Gly Asn Glu Val Ser Arg Ala Val Leu Leu Pro GlnSer 260 265 270 tgc taa 822 Cys 30 273 PRT Homo sapiens human HLA-DObeta 30 Met Gly Ser Gly Trp Val Pro Trp Val Val Ala Leu Leu Val Asn Leu1 5 10 15 Thr Gln Leu Asp Ser Ser Met Thr Gln Gly Thr Asp Ser Pro GluAsp 20 25 30 Phe Val Ile Gln Ala Lys Ala Asp Cys Tyr Phe Thr Asn Gly ThrGlu 35 40 45 Lys Val Gln Phe Val Val Arg Phe Ile Phe Asn Leu Glu Glu TyrVal 50 55 60 Arg Phe Asp Ser Asp Val Gly Met Phe Val Ala Leu Thr Lys LeuGly 65 70 75 80 Gln Pro Asp Ala Glu Gln Trp Asn Ser Arg Leu Asp Leu LeuGlu Arg 85 90 95 Ser Arg Gln Ala Val Asp Gly Val Cys Arg His Asn Tyr ArgLeu Gly 100 105 110 Ala Pro Phe Thr Val Gly Arg Lys Val Gln Pro Glu ValThr Val Tyr 115 120 125 Pro Glu Arg Thr Pro Leu Leu His Gln His Asn LeuLeu His Cys Ser 130 135 140 Val Thr Gly Phe Tyr Pro Gly Asp Ile Lys IleLys Trp Phe Leu Asn 145 150 155 160 Gly Gln Glu Glu Arg Ala Gly Val MetSer Thr Gly Pro Ile Arg Asn 165 170 175 Gly Asp Trp Thr Phe Gln Thr ValVal Met Leu Glu Met Thr Pro Glu 180 185 190 Leu Gly His Val Tyr Thr CysLeu Val Asp His Ser Ser Leu Leu Ser 195 200 205 Pro Val Ser Val Glu TrpArg Ala Gln Ser Glu Tyr Ser Trp Arg Lys 210 215 220 Met Leu Ser Gly IleAla Ala Phe Leu Leu Gly Leu Ile Phe Leu Leu 225 230 235 240 Val Gly IleVal Ile Gln Leu Arg Ala Gln Lys Gly Tyr Val Arg Thr 245 250 255 Gln MetSer Gly Asn Glu Val Ser Arg Ala Val Leu Leu Pro Gln Ser 260 265 270 Cys31 700 DNA Homo sapiens CDS (1)...(681) human MB-1 Ig-alpha 31 atg cctggg ggt cca gga gtc ctc caa gct ctg cct gcc acc atc ttc 48 Met Pro GlyGly Pro Gly Val Leu Gln Ala Leu Pro Ala Thr Ile Phe 1 5 10 15 ctc ctcttc ctg ctg tct gct gtc tac ctg ggc cct ggg tgc cag gcc 96 Leu Leu PheLeu Leu Ser Ala Val Tyr Leu Gly Pro Gly Cys Gln Ala 20 25 30 ctg tgg atgcac aag gtc cca gca tca ttg atg gtg agc ctg ggg gaa 144 Leu Trp Met HisLys Val Pro Ala Ser Leu Met Val Ser Leu Gly Glu 35 40 45 gac gcc cac ttccaa tgc ccg cac aat agc agc aac aac gcc aac gtc 192 Asp Ala His Phe GlnCys Pro His Asn Ser Ser Asn Asn Ala Asn Val 50 55 60 acc tgg tgg cgc gtcctc cat ggc aac tac acg tgg ccc cct gag ttc 240 Thr Trp Trp Arg Val LeuHis Gly Asn Tyr Thr Trp Pro Pro Glu Phe 65 70 75 80 ttg ggc ccg ggc gaggac ccc aat ggt acg ctg atc atc cag aat gtg 288 Leu Gly Pro Gly Glu AspPro Asn Gly Thr Leu Ile Ile Gln Asn Val 85 90 95 aac aag agc cat ggg ggcata tac gtg tgc cgg gtc cag gag ggc aac 336 Asn Lys Ser His Gly Gly IleTyr Val Cys Arg Val Gln Glu Gly Asn 100 105 110 gag tca tac cag cag tcctgc ggc acc tac ctc cgc gtg cgc cag ccg 384 Glu Ser Tyr Gln Gln Ser CysGly Thr Tyr Leu Arg Val Arg Gln Pro 115 120 125 ccc ccc agg ccc ttc ctggac atg ggg gag ggc acc aag aac cga atc 432 Pro Pro Arg Pro Phe Leu AspMet Gly Glu Gly Thr Lys Asn Arg Ile 130 135 140 atc aca gcc gag ggg atcatc ctc ctg ttc tgc gcg gtg gtg cct ggg 480 Ile Thr Ala Glu Gly Ile IleLeu Leu Phe Cys Ala Val Val Pro Gly 145 150 155 160 acg ctg ctg ctg ttcagg aaa cga tgg cag aac gag aag ctc ggg ttg 528 Thr Leu Leu Leu Phe ArgLys Arg Trp Gln Asn Glu Lys Leu Gly Leu 165 170 175 gat gcc ggg gat gaatat gaa gat gaa aac ctt tat gaa ggc ctg aac 576 Asp Ala Gly Asp Glu TyrGlu Asp Glu Asn Leu Tyr Glu Gly Leu Asn 180 185 190 ctg gac gac tgc tccatg tat gag gac atc tcc cgg ggc ctc cag ggc 624 Leu Asp Asp Cys Ser MetTyr Glu Asp Ile Ser Arg Gly Leu Gln Gly 195 200 205 acc tac cag gat gtgggc agc ctc aac ata gga gat gtc cag ctg gag 672 Thr Tyr Gln Asp Val GlySer Leu Asn Ile Gly Asp Val Gln Leu Glu 210 215 220 aag ccg tgacacccctactcctgcca gg 700 Lys Pro 225 32 226 PRT Homo sapiens human MB-1Ig-alpha 32 Met Pro Gly Gly Pro Gly Val Leu Gln Ala Leu Pro Ala Thr IlePhe 1 5 10 15 Leu Leu Phe Leu Leu Ser Ala Val Tyr Leu Gly Pro Gly CysGln Ala 20 25 30 Leu Trp Met His Lys Val Pro Ala Ser Leu Met Val Ser LeuGly Glu 35 40 45 Asp Ala His Phe Gln Cys Pro His Asn Ser Ser Asn Asn AlaAsn Val 50 55 60 Thr Trp Trp Arg Val Leu His Gly Asn Tyr Thr Trp Pro ProGlu Phe 65 70 75 80 Leu Gly Pro Gly Glu Asp Pro Asn Gly Thr Leu Ile IleGln Asn Val 85 90 95 Asn Lys Ser His Gly Gly Ile Tyr Val Cys Arg Val GlnGlu Gly Asn 100 105 110 Glu Ser Tyr Gln Gln Ser Cys Gly Thr Tyr Leu ArgVal Arg Gln Pro 115 120 125 Pro Pro Arg Pro Phe Leu Asp Met Gly Glu GlyThr Lys Asn Arg Ile 130 135 140 Ile Thr Ala Glu Gly Ile Ile Leu Leu PheCys Ala Val Val Pro Gly 145 150 155 160 Thr Leu Leu Leu Phe Arg Lys ArgTrp Gln Asn Glu Lys Leu Gly Leu 165 170 175 Asp Ala Gly Asp Glu Tyr GluAsp Glu Asn Leu Tyr Glu Gly Leu Asn 180 185 190 Leu Asp Asp Cys Ser MetTyr Glu Asp Ile Ser Arg Gly Leu Gln Gly 195 200 205 Thr Tyr Gln Asp ValGly Ser Leu Asn Ile Gly Asp Val Gln Leu Glu 210 215 220 Lys Pro 225 33723 DNA Homo sapiens CDS (17)...(706) human Ig-beta protein 33gaattccgcg gtgacc atg gcc agg ctg gcg ttg tct cct gtg ccc agc cac 52 MetAla Arg Leu Ala Leu Ser Pro Val Pro Ser His 1 5 10 tgg atg gtg gcg ttgctg ctg ctg ctc tca gct gag cca gta cca gca 100 Trp Met Val Ala Leu LeuLeu Leu Leu Ser Ala Glu Pro Val Pro Ala 15 20 25 gcc aga tcg gag gac cggtac cgg aat ccc aaa ggt agt gct tgt tcg 148 Ala Arg Ser Glu Asp Arg TyrArg Asn Pro Lys Gly Ser Ala Cys Ser 30 35 40 cgg atc tgg cag agc cca cgtttc ata gcc agg aaa cgg cgc ttc acg 196 Arg Ile Trp Gln Ser Pro Arg PheIle Ala Arg Lys Arg Arg Phe Thr 45 50 55 60 gtg aaa atg cac tgc tac atgaac agc gcc tcc ggc aat gtg agc tgg 244 Val Lys Met His Cys Tyr Met AsnSer Ala Ser Gly Asn Val Ser Trp 65 70 75 ctc tgg aag cag gag atg gac gagaat ccc cag cag ctg aag ctg gaa 292 Leu Trp Lys Gln Glu Met Asp Glu AsnPro Gln Gln Leu Lys Leu Glu 80 85 90 aag ggc cgc atg gaa gag tcc cag aacgaa tct ctc gcc acc ctc acc 340 Lys Gly Arg Met Glu Glu Ser Gln Asn GluSer Leu Ala Thr Leu Thr 95 100 105 atc caa ggc atc cgg ttt gag gac aatggc atc tac ttc tgc cag cag 388 Ile Gln Gly Ile Arg Phe Glu Asp Asn GlyIle Tyr Phe Cys Gln Gln 110 115 120 aag tgc aac aac acc tcg gag gtc taccag ggc tgc ggc aca gag ctg 436 Lys Cys Asn Asn Thr Ser Glu Val Tyr GlnGly Cys Gly Thr Glu Leu 125 130 135 140 cga gtc atg gga ttc agc acc ttggca cag ctg aag cag agg aac acg 484 Arg Val Met Gly Phe Ser Thr Leu AlaGln Leu Lys Gln Arg Asn Thr 145 150 155 ctg aag gat ggt atc atc atg atccag acg ctg ctg atc atc ctc ttc 532 Leu Lys Asp Gly Ile Ile Met Ile GlnThr Leu Leu Ile Ile Leu Phe 160 165 170 atc atc gtg cct atc ttc ctg ctgctg gac aag gat gac agc aag gct 580 Ile Ile Val Pro Ile Phe Leu Leu LeuAsp Lys Asp Asp Ser Lys Ala 175 180 185 ggc atg gag gaa gat cac acc tacgag ggc ctg gac att gac cag aca 628 Gly Met Glu Glu Asp His Thr Tyr GluGly Leu Asp Ile Asp Gln Thr 190 195 200 gcc acc tat gag gac ata gtg acgctg cgg aca ggg gaa gtg aag tgg 676 Ala Thr Tyr Glu Asp Ile Val Thr LeuArg Thr Gly Glu Val Lys Trp 205 210 215 220 tct gta ggt gag cac cca ggccag gag tgagagccag gtcgccccat 723 Ser Val Gly Glu His Pro Gly Gln Glu225 34 229 PRT Homo sapiens human Ig-beta protein 34 Met Ala Arg Leu AlaLeu Ser Pro Val Pro Ser His Trp Met Val Ala 1 5 10 15 Leu Leu Leu LeuLeu Ser Ala Glu Pro Val Pro Ala Ala Arg Ser Glu 20 25 30 Asp Arg Tyr ArgAsn Pro Lys Gly Ser Ala Cys Ser Arg Ile Trp Gln 35 40 45 Ser Pro Arg PheIle Ala Arg Lys Arg Arg Phe Thr Val Lys Met His 50 55 60 Cys Tyr Met AsnSer Ala Ser Gly Asn Val Ser Trp Leu Trp Lys Gln 65 70 75 80 Glu Met AspGlu Asn Pro Gln Gln Leu Lys Leu Glu Lys Gly Arg Met 85 90 95 Glu Glu SerGln Asn Glu Ser Leu Ala Thr Leu Thr Ile Gln Gly Ile 100 105 110 Arg PheGlu Asp Asn Gly Ile Tyr Phe Cys Gln Gln Lys Cys Asn Asn 115 120 125 ThrSer Glu Val Tyr Gln Gly Cys Gly Thr Glu Leu Arg Val Met Gly 130 135 140Phe Ser Thr Leu Ala Gln Leu Lys Gln Arg Asn Thr Leu Lys Asp Gly 145 150155 160 Ile Ile Met Ile Gln Thr Leu Leu Ile Ile Leu Phe Ile Ile Val Pro165 170 175 Ile Phe Leu Leu Leu Asp Lys Asp Asp Ser Lys Ala Gly Met GluGlu 180 185 190 Asp His Thr Tyr Glu Gly Leu Asp Ile Asp Gln Thr Ala ThrTyr Glu 195 200 205 Asp Ile Val Thr Leu Arg Thr Gly Glu Val Lys Trp SerVal Gly Glu 210 215 220 His Pro Gly Gln Glu 225 35 5053 DNA ArtificialSequence vector pEP2 35 gacggatcgg gagatctccc gatcccctat ggtcgactctcagtacaatc tgctctgatg 60 ccgcatagtt aagccagtat ctgctccctg cttgtgtgttggaggtcgct gagtagtgcg 120 cgagcaaaat ttaagctaca acaaggcaag gcttgaccgacaattgcatg aagaatctgc 180 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggccagatatacg cgttgacatt 240 gattattgac tagttattaa tagtaatcaa ttacggggtcattagttcat agcccatata 300 tggagttccg cgttacataa cttacggtaa atggcccgcctggctgaccg cccaacgacc 360 cccgcccatt gacgtcaata atgacgtatg ttcccatagtaacgccaata gggactttcc 420 attgacgtca atgggtggac tatttacggt aaactgcccacttggcagta catcaagtgt 480 atcatatgcc aagtacgccc cctattgacg tcaatgacggtaaatggccc gcctggcatt 540 atgcccagta catgacctta tgggactttc ctacttggcagtacatctac gtattagtca 600 tcgctattac catggtgatg cggttttggc agtacatcaatgggcgtgga tagcggtttg 660 actcacgggg atttccaagt ctccacccca ttgacgtcaatgggagtttg ttttggcacc 720 aaaatcaacg ggactttcca aaatgtcgta acaactccgccccattgacg caaatgggcg 780 gtaggcgtgt acggtgggag gtctatataa gcagagctctctggctaact agagaaccca 840 ctgcttactg gcttatcgaa attaatacga ctcactatagggagacccaa gctggctaga 900 gtaagtaccg cctatagagt ctataggccc acccccttggcttcttatgc atgctatact 960 gtttttggct tggggtctat acacccccgc ttcctcatgttataggtgat ggtatagctt 1020 agcctatagg tgtgggttat tgaccattat tgaccactcccctattggtg acgatacttt 1080 ccattactaa tccataacat ggctctttgc cacaactctctttattggct atatgccaat 1140 acactgtcct tcagagactg acacggactc tgtatttttacaggatgggg tctcatttat 1200 tatttacaaa ttcacatata caacaccacc gtccccagtgcccgcagttt ttattaaaca 1260 taacgtggga tctccacgcg aatctcgggt acgtgttccggacatgggct cttctccggt 1320 agcggcggag cttctacatc cgagccctgc tcccatgcctccagcgactc atggtcgctc 1380 ggcagctcct tgctcctaac agtggaggcc agacttaggcacagcacgat gcccaccacc 1440 accagtgtgc cgcacaaggc cgtggcggta gggtatgtgtctgaaaatga gctcggggag 1500 cgggcttgca ccgctgacgc atttggaaga cttaaggcagcggcagaaga agatgcaggc 1560 agctgagttg ttgtgttctg ataagagtca gaggtaactcccgttgcggt gctgttaacg 1620 gtggagggca gtgtagtctg agcagtactc gttgctgccgcgcgcgccac cagacataat 1680 agctgacaga ctaacagact gttcctttcc atgggtcttttctgcaggct agccggcctg 1740 aattcggata tccaagcttg atgaataaaa gatcagagctctagtgatct gtgtgttggt 1800 tttttgtgtg ctcgagcccc agctggttct ttccgcctcagaagccatag agcccaccgc 1860 atccccagca tgcctgctat tgtcttccca atcctcccccttgctgtcct gccccacccc 1920 accccccaga atagaatgac acctactcag acaatgcgatgcaatttcct cattttatta 1980 ggaaaggaca gtgggagtgg caccttccag ggtcaaggaaggcacggggg aggggcaaac 2040 aacagatggc tggcaactag aaggcacagt cgaggctgatcagcgagctc tagcggtacc 2100 ggcattagtc tatggccgac tctagatttt ctccttgcggccgccctaga tgcatgctcg 2160 atcgacctgc agttggacct gggagtggac acctgtggagagaaaggcaa agtggatgtc 2220 attgtcactc aagtgtatgg ccagatctca agcctgccacacctcaagct agcttgacaa 2280 caaaaagatt gtcttttctg accagatgga cgcggccaccctcaaaggca tcaccgcggg 2340 ccaggtgaat atcaaatcct cctcgttttt ggaaactgacaatcttagcg cagaagtcat 2400 gcccgctttt gagagggagt actcacccca acagctggccctcgcagaca gcgaattaat 2460 tccagcacac tggcggccgt tactagtgga tccgagctcgcaagctagct tgggtctccc 2520 tatagtgagt cgtattaatt tcgataagcc agtaagcagtgggttctcta gttagccaga 2580 gagctctgct tatatagacc tcccaccgta cacgcctaccgcccatttgc gtcaatgggg 2640 cggagttgtt acgacatttt ggaaagtccc gttgattttggtgccaaaac aaactcccat 2700 tgacgtcaat ggggtggaga cttggaaatc cccgtgagtcaaaccgctat ccacgcccat 2760 tgatgtactg ccaaaaccgc atcaccatgg taatagcgatgactaatacg tagatgtact 2820 gccaagtagg aaagtcccat aaggtcatgt actgggcataatgccaggcg ggccatttac 2880 cgtcattgac gtcaataggg ggcgtacttg gcatatgatacacttgatgt actgccaagt 2940 gggcagttta ccgtaaatag tccacccatt gacgtcaatggaaagtccct attggcgtta 3000 ctatgggaac atacgtcatt attgacgtca atgggcgggggtcgttgggc ggtcagccag 3060 gcgggccatt taccgtaagt tatgtaacgc ggaactccatatatgggcta tgaactaatg 3120 accccgtaat tgattactat taataactag tcaataatcaatgtcctgca ttaatgaatc 3180 ggccaacgcg cggggagagg cggtttgcgt attgggcgctcttccgcttc ctcgctcact 3240 gactcgctgc gctcggtcgt tcggctgcgg cgagcggtatcagctcactc aaaggcggta 3300 atacggttat ccacagaatc aggggataac gcaggaaagaacatgtgagc aaaaggccag 3360 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgtttttccatag gctccgcccc 3420 cctgacgagc atcacaaaaa tcgacgctca agtcagaggtggcgaaaccc gacaggacta 3480 taaagatacc aggcgtttcc ccctggaagc tccctcgtgcgctctcctgt tccgaccctg 3540 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaagcgtggcgct ttctcaatgc 3600 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgctccaagctggg ctgtgtgcac 3660 gaaccccccg ttcagcccga ccgctgcgcc ttatccggtaactatcgtct tgagtccaac 3720 ccggtaagac acgacttatc gccactggca gcagccactggtaacaggat tagcagagcg 3780 aggtatgtag gcggtgctac agagttcttg aagtggtggcctaactacgg ctacactaga 3840 aggacagtat ttggtatctg cgctctgctg aagccagttaccttcggaaa aagagttggt 3900 agctcttgat ccggcaaaca aaccaccgct ggtagcggtggtttttttgt ttgcaagcag 3960 cagattacgc gcagaaaaaa aggatctcaa gaagatcctttgatcttttc tacggggtct 4020 gacgctcagt ggaacgaaaa ctcacgttaa gggattttggtcatgaacaa taaaactgtc 4080 tgcttacata aacagtaata caaggggtgt tatgagccatattcaacggg aaacgtcttg 4140 ctcgaggccg cgattaaatt ccaacatgga tgctgatttatatgggtata aatgggctcg 4200 cgataatgtc gggcaatcag gtgcgacaat ctatcgattgtatgggaagc ccgatgcgcc 4260 agagttgttt ctgaaacatg gcaaaggtag cgttgccaatgatgttacag atgagatggt 4320 cagactaaac tggctgacgg aatttatgcc tcttccgaccatcaagcatt ttatccgtac 4380 tcctgatgat gcatggttac tcaccactgc gatccccgggaaaacagcat tccaggtatt 4440 agaagaatat cctgattcag gtgaaaatat tgttgatgcgctggcagtgt tcctgcgccg 4500 gttgcattcg attcctgttt gtaattgtcc ttttaacagcgatcgcgtat ttcgtctcgc 4560 tcaggcgcaa tcacgaatga ataacggttt ggttgatgcgagtgattttg atgacgagcg 4620 taatggctgg cctgttgaac aagtctggaa agaaatgcataaacttttgc cattctcacc 4680 ggattcagtc gtcactcatg gtgatttctc acttgataaccttatttttg acgaggggaa 4740 attaataggt tgtattgatg ttggacgagt cggaatcgcagaccgatacc aggatcttgc 4800 catcctatgg aactgcctcg gtgagttttc tccttcattacagaaacggc tttttcaaaa 4860 atatggtatt gataatcctg atatgaataa attgcagtttcatttgatgc tcgatgagtt 4920 tttctaatca gaattggtta attggttgta acactggcagagcatcatga gcggatacat 4980 atttgaatgt atttagaaaa ataaacaaat aggggttccgcgcacatttc cccgaaaagt 5040 gccacctgac gtc 5053 36 411 DNA ArtificialSequence vector pMIN.0 36 gctagcgccg ccacc atg gga atg cag gtg cag atccag agc ctg ttt ctg 51 Met Gly Met Gln Val Gln Ile Gln Ser Leu Phe Leu 15 10 ctc ctc ctg tgg gtg ccc ggg tcc aga gga cac acc ctg tgg aag gcc 99Leu Leu Leu Trp Val Pro Gly Ser Arg Gly His Thr Leu Trp Lys Ala 15 20 25gga atc ctg tat aag gcc aag ttc gtg gct gcc tgg acc ctg aag gct 147 GlyIle Leu Tyr Lys Ala Lys Phe Val Ala Ala Trp Thr Leu Lys Ala 30 35 40 gccgct ttc ctg cct agc gat ttc ttt cct agc gtg aag ctg acc cca 195 Ala AlaPhe Leu Pro Ser Asp Phe Phe Pro Ser Val Lys Leu Thr Pro 45 50 55 60 ctgtgc gtg acc ctg tat atg gat gac gtg gtg ctg gga gcc agc atc 243 Leu CysVal Thr Leu Tyr Met Asp Asp Val Val Leu Gly Ala Ser Ile 65 70 75 atc aacttc gag aag ctg gga ctg tcc aga tac gtg gct agg ctg atc 291 Ile Asn PheGlu Lys Leu Gly Leu Ser Arg Tyr Val Ala Arg Leu Ile 80 85 90 ctg aag gagcct gtg cac ggc gtg tcc acc ctg cca gag acc acc gtg 339 Leu Lys Glu ProVal His Gly Val Ser Thr Leu Pro Glu Thr Thr Val 95 100 105 gtg agg aggacc gtg tac tat gga gtg cct gtg tgg aag tgg ctg agc 387 Val Arg Arg ThrVal Tyr Tyr Gly Val Pro Val Trp Lys Trp Leu Ser 110 115 120 ctg ctg gtgccc ttt gtg ggt acc 411 Leu Leu Val Pro Phe Val Gly Thr 125 130 37 132PRT Artificial Sequence vector pMIN.0 37 Met Gly Met Gln Val Gln Ile GlnSer Leu Phe Leu Leu Leu Leu Trp 1 5 10 15 Val Pro Gly Ser Arg Gly HisThr Leu Trp Lys Ala Gly Ile Leu Tyr 20 25 30 Lys Ala Lys Phe Val Ala AlaTrp Thr Leu Lys Ala Ala Ala Phe Leu 35 40 45 Pro Ser Asp Phe Phe Pro SerVal Lys Leu Thr Pro Leu Cys Val Thr 50 55 60 Leu Tyr Met Asp Asp Val ValLeu Gly Ala Ser Ile Ile Asn Phe Glu 65 70 75 80 Lys Leu Gly Leu Ser ArgTyr Val Ala Arg Leu Ile Leu Lys Glu Pro 85 90 95 Val His Gly Val Ser ThrLeu Pro Glu Thr Thr Val Val Arg Arg Thr 100 105 110 Val Tyr Tyr Gly ValPro Val Trp Lys Trp Leu Ser Leu Leu Val Pro 115 120 125 Phe Val Gly Thr130 38 390 DNA Artificial Sequence vector pMIN.1 38 gctagcgccg ccacc atggga atg cag gtg cag atc cag agc ctg ttt ctg 51 Met Gly Met Gln Val GlnIle Gln Ser Leu Phe Leu 1 5 10 ctc ctc ctg tgg gtg ccc ggg tcc aga ggacac acc ctg tgg aag gcc 99 Leu Leu Leu Trp Val Pro Gly Ser Arg Gly HisThr Leu Trp Lys Ala 15 20 25 gga atc ctg tat aag gcc aag ttc gtg gct gcctgg acc ctg aag gct 147 Gly Ile Leu Tyr Lys Ala Lys Phe Val Ala Ala TrpThr Leu Lys Ala 30 35 40 gcc gct ttc ctg cct agc gat ttc ttt cct agc gtgaag ctg acc cca 195 Ala Ala Phe Leu Pro Ser Asp Phe Phe Pro Ser Val LysLeu Thr Pro 45 50 55 60 ctg tgc gtg acc ctg tat atg gat gac gtg gtg ctggga gtg gga ctg 243 Leu Cys Val Thr Leu Tyr Met Asp Asp Val Val Leu GlyVal Gly Leu 65 70 75 tcc agg tac gtg gct agg ctg atc ctg aag gag cct gtgcac ggc gtg 291 Ser Arg Tyr Val Ala Arg Leu Ile Leu Lys Glu Pro Val HisGly Val 80 85 90 tcc acc ctg cca gag acc acc gtg gtg agg agg acc gtg tactat gga 339 Ser Thr Leu Pro Glu Thr Thr Val Val Arg Arg Thr Val Tyr TyrGly 95 100 105 gtg cct gtg tgg aag tgg ctg agc ctg ctg gtg ccc ttt gtg381 Val Pro Val Trp Lys Trp Leu Ser Leu Leu Val Pro Phe Val 110 115 120tgaggtacc 390 39 122 PRT Artificial Sequence vector pMIN.1 39 Met GlyMet Gln Val Gln Ile Gln Ser Leu Phe Leu Leu Leu Leu Trp 1 5 10 15 ValPro Gly Ser Arg Gly His Thr Leu Trp Lys Ala Gly Ile Leu Tyr 20 25 30 LysAla Lys Phe Val Ala Ala Trp Thr Leu Lys Ala Ala Ala Phe Leu 35 40 45 ProSer Asp Phe Phe Pro Ser Val Lys Leu Thr Pro Leu Cys Val Thr 50 55 60 LeuTyr Met Asp Asp Val Val Leu Gly Val Gly Leu Ser Arg Tyr Val 65 70 75 80Ala Arg Leu Ile Leu Lys Glu Pro Val His Gly Val Ser Thr Leu Pro 85 90 95Glu Thr Thr Val Val Arg Arg Thr Val Tyr Tyr Gly Val Pro Val Trp 100 105110 Lys Trp Leu Ser Leu Leu Val Pro Phe Val 115 120 40 36 DNA ArtificialSequence primer oligonucleotide murIi-F 40 gctagcgccg ccaccatggatgaccaacgc gacctc 36 41 27 DNA Artificial Sequence primeroligonucleotide murIi-R 41 ggtacctcac agggtgactt gacccag 27 42 42 DNAArtificial Sequence primer oligonucleotide IiPADRE-R 42 cagggtccaggcagccacga acttggccac aggtttggca ga 42 43 43 DNA Artificial Sequenceprimer oligonucleotide IiPADRE-F 43 ggctgcctgg accctgaagg ctgccgctatgtccatggat aac 43 44 70 DNA Artificial Sequence oligo 1 44 cttcgcatgaagcttatcag ccaggctgtg cacgccgctc acgccgaaat caacgaagct 60 ggaagaaccc 7045 70 DNA Artificial Sequence oligo 2 45 ttctggtcag cagaaagaacaggataggag cgtttggagg gcgataagct ggaggggttc 60 ttccagcttc 70 46 71 DNAArtificial Sequence oligo 3 46 ttctgctgac cagaatcctg acaatcccccagtccctgga cgccaagttc gtggctgcct 60 ggaccctgaa g 71 47 34 DNA ArtificialSequence Help-epR primer 47 ggtacctcaa gcggcagcct tcagggtcca ggca 34 4817 PRT Artificial Sequence residues 323-339 of ovalbumin (Ova323-339) 48Ile Ser Gln Ala Val His Ala Ala His Ala Glu Ile Asn Glu Ala Gly 1 5 1015 Arg 49 13 PRT Artificial Sequence residues 128-141 of HBV coreantigen (HBVcore 128) 49 Thr Pro Pro Ala Tyr Arg Pro Pro Asn Ala Pro IleLeu 1 5 10 50 15 PRT Artificial Sequence residues 182-196 of HBV env(HBVenv182) 50 Phe Phe Leu Leu Thr Arg Ile Leu Thr Ile Pro Gln Ser LeuAsp 1 5 10 15 51 19 DNA Artificial Sequence Th-Pad-R primer 51agcggcagcc ttcagggtc 19 52 43 DNA Artificial Sequence IiPADRE-F primer52 ggctgcctgg accctgaagg ctgccgctat gtccatggat aac 43 53 20 DNAArtificial Sequence primer Th-ova-F 53 atcagccagg ctgtgcacgc 20 54 27DNA Artificial Sequence primer 1F oligonucleotide KappaSig-F 54gctagcgccg ccaccatggg aatgcag 27 55 27 DNA Artificial Sequence primer 1Roligonucleotide Kappa-Th-R 55 cacagcctgg ctgattcctc tggaccc 27 56 33 DNAArtificial Sequence primer 2F oligonucleotide PAD/LAMP-F 56 ctgaaggctgccgctaacaa catgttgatc ccc 33 57 24 DNA Artificial Sequence primer 2Roligonucleotide LAMP-CYTOR 57 ggtaccctag atggtctgat agcc 24 58 33 DNAArtificial Sequence primer 1F oligonucleotide H2-Mb-1F 58 gccgctagcgccgccaccat ggctgcactc tgg 33 59 30 DNA Artificial Sequence primer 1Roligonucleotide H2-Mb-1R 59 cacagcctgg ctgatcccca tacagtgcag 30 60 30DNA Artificial Sequence primer 2F oligonucleotide H2-Mb-2F 60 ctgaaggctgccgctaaggt ctctgtgtct 30 61 24 DNA Artificial Sequence primer 2Roligonucleotide H2-Mb-2R 61 gcgggtaccc taatgccgtc cttc 24 62 33 DNAArtificial Sequence primer 1F oligonucleotide H2-Ob-1F 62 gcggctagcgccgccaccat gggcgctggg agg 33 63 30 DNA Artificial Sequence primer 1Roligonucleotide H2-Ob-1R 63 tgcacagcct ggctgatgga atccagcctc 30 64 30DNA Artificial Sequence primer 2F oligonucleotide H2-Ob-2F 64 ctgaaggctgccgctatact gagtggagct 30 65 26 DNA Artificial Sequence primer 2Roligonucleotide H2-Ob-2R 65 gccggtacct catgtgacat gtcccg 26 66 80 DNAArtificial Sequence PADRE-Influenza matrix 5′ primer 66 gctagcgccgccaccatggc caagttcgtg gctgcctgga ccctgaaggc tgccgctatg 60 agtcttctaaccgaggtcga 80 67 30 DNA Artificial Sequence PADRE-Influenza matrix 3′primer 67 tcacttgaat cgctgcatct gcacccccat 30 68 58 DNA ArtificialSequence PADRE-HBV-s one oligonucleotide 68 gctagcgccg ccaccatggccaagttcgtg gctgcctgga ccctgaaggc tgccgctc 58 69 58 DNA ArtificialSequence PADRE-HBV-s second oligonucleotide 69 ctcgagagcg gcagccttcagggtccaggc agccacgaac ttggccatgg tggcggcg 58 70 33 DNA ArtificialSequence primer 1F oligonucleotide Ig alpha-1F 70 gcggctagcg ccgccaccatgccagggggt cta 33 71 30 DNA Artificial Sequence primer 1Roligonucleotide Ig alpha-1R 71 gcacagcctg gctgatggcc tggcatccgg 30 72 30DNA Artificial Sequence primer 2F oligonucleotide Ig alpha-2F 72ctgaaggctg ccgctgggat catcttgctg 30 73 27 DNA Artificial Sequence primer2R oligonucleotide Ig alpha-2R 73 gcgggtacct catggctttt ccagctg 27 74 33DNA Artificial Sequence primer 1F oligonucleotide B29-1F 74 gcggctagcgccgccaccat ggccacactg gtg 33 75 30 DNA Artificial Sequence primer 1Roligonucleotide B29-1R 75 cacagcctgg ctgatcggct cacctgagaa 30 76 30 DNAArtificial Sequence primer 2F oligonucleotide B29-2F (30mer) 76ctgaaggctg ccgctattat cttgatccag 30 77 27 DNA Artificial Sequence primer2R oligonucleotide B29-2R (27mer) 77 gccggtacct cattcctggc ctggatg 27 7821 DNA Artificial Sequence pUC4K amplification primer 78 tctgatgttacattgcacaa g 21 79 33 DNA Artificial Sequence pUC4K amplification primer79 gcgcactcat gatgctctgc cagtgttaca acc 33 80 30 DNA Artificial SequenceCMV DNA amplification primer 80 gcgtctagag taagtaccgc ctatagactc 30 8130 DNA Artificial Sequence CMV DNA amplification primer 81 ccggctagcctgcagaaaag acccatggaa 30 82 49 DNA Artificial Sequence polylinker oligo82 ggccgcaagg aaaaaatcta gagtcggcca tagactaatg ccggtaccg 49 83 50 DNAArtificial Sequence polylinker oligo 83 ctagcggtac cggcattagt ctatggcccgactctagatt ttttccttgc 50 84 72 DNA Artificial Sequence oligo annealed toproduce fragment with polylinker, polyadenylation signal and EcoRI andXhoI cohesive ends 84 aattcggata tccaagcttg atgaataaaa gatcagagctctagtgatct gtgtgttggt 60 ttttttgtgt gc 72 85 72 DNA Artificial Sequenceoligo annealed to produce fragment with polylinker, polyadenylationsignal and EcoRI and XhoI cohesive ends 85 tcgagcacac aaaaaaccaacacacagatc actagagctc tgatcttttt attcatcaag 60 cttggatatc cg 72 86 20PRT Artificial Sequence consensus mouse Ig Kappa signal sequence 86 MetGln Val Gln Ile Gln Ser Leu Phe Leu Leu Leu Leu Trp Val Pro 1 5 10 15Gly Ser Arg Gly 20 87 60 DNA Artificial Sequence nucleotides encodingconsensus mouse Ig Kappa signal sequence 87 atgcaggtgc agatccagagcctgtttctg ctcctcctgt gggtgcccgg gtccagagga 60 88 11 PRT ArtificialSequence HBV pol 149-159 (A11 restricted) (peptide 1147.16) 88 His ThrLeu Trp Lys Ala Gly Ile Leu Tyr Lys 1 5 10 89 33 DNA Artificial Sequencenucleotides encoding HBV pol 149-159 (A11 restricted) 89 cacaccctgtggaaggccgg aatcctgtat aag 33 90 39 DNA Artificial Sequence nucleotidesencoding PADRE-universal MHC class II epitope 90 gccaagttcg tggctgcctggaccctgaag gctgccgct 39 91 10 PRT Artificial Sequence HBV core 18-27 (A2restricted) (peptide 924.07) 91 Phe Leu Pro Ser Asp Phe Phe Pro Ser Val1 5 10 92 30 DNA Artificial Sequence nucleotides encoding HBV core 18-27(A2 restricted) 92 ttcctgccta gcgatttctt tcctagcgtg 30 93 9 PRTArtificial Sequence HIV env 120-128 (A2 restricted) (peptide 1211.04) 93Lys Leu Thr Pro Leu Cys Val Thr Leu 1 5 94 27 DNA Artificial Sequencenucleotides encoding HIV env 120-128 (A2 restricted) 94 aagctgaccccactgtgcgt gaccctg 27 95 9 PRT Artificial Sequence HBV pol 551-559 (A2restricted) (peptide 1090.14) 95 Tyr Met Asp Asp Val Val Leu Gly Ala 1 596 27 DNA Artificial Sequence nucleotides encoding HBV pol 551-559 (A2restricted) 96 tatatggatg acgtggtgct gggagcc 27 97 8 PRT ArtificialSequence mouse ovalbumin 257-264 (Kb restricted) 97 Ser Ile Ile Asn PheGlu Lys Leu 1 5 98 24 DNA Artificial Sequence nucleotides encoding mouseovalbumin 257-264 (Kb restricted) 98 agcatcatca acttcgagaa gctg 24 99 9PRT Artificial Sequence HBV pol 455-463 (A2 restricted) (peptide1168.02) 99 Gly Leu Ser Arg Tyr Val Ala Arg Leu 1 5 100 27 DNAArtificial Sequence nucleotides encoding HBV pol 455-463 (A2 restricted)100 ggactgttca gatacgtggc taggctg 27 101 9 PRT Artificial Sequence HIVpol 476-484 (A2 restricted) (peptide 941.031) 101 Ile Leu Lys Glu ProVal His Gly Val 1 5 102 27 DNA Artificial Sequence nucleotides encodingHIV pol 476-484 (A2 restricted) 102 atcctgaagg agcctgtgca cggcgtg 27 10311 PRT Artificial Sequence HBV core 141-151 (A11 restricted) (peptide1083.01) 103 Ser Thr Leu Pro Glu Thr Thr Val Val Arg Arg 1 5 10 104 33DNA Artificial Sequence nucleotides encoding HBV core 141-151 (A11restricted) 104 tccaccctgc cagagaccac cgtggtgagg aga 33 105 10 PRTArtificial Sequence HIV env 49-58 (A11 restricted) (peptide 1069.43) 105Thr Val Tyr Tyr Gly Val Pro Val Trp Lys 1 5 10 106 30 DNA ArtificialSequence nucleotides encoding HIV env 49-58 (A11 restricted) 106accgtgtact atggagtgcc tgtgtggaag 30 107 9 PRT Artificial SequenceHBVadr-ENV (S Ag 335-343) (A2 restricted) (peptide 1013.0102) 107 TrpLeu Ser Leu Leu Val Pro Phe Val 1 5 108 27 DNA Artificial Sequencenucleotides encoding HBV env 335-343 (A2 restricted) 108 tggctgagcctgctggtgcc ctttgtg 27 109 12 DNA Artificial Sequence consensus Kozaksequence 109 gccgccacca tg 12 110 70 DNA Artificial Sequence Min1 oligo110 gaggagcaga aacaggctct ggatctgcac ctgcattccc atggtggcgg cgctagcaag 60cttcttgcgc 70 111 71 DNA Artificial Sequence Min2 oligo 111 cctgtttctgctcctcctgt gggtgcccgg gtccagagga cacaccctgt ggaaggccgg 60 aatcctgtat a71 112 70 DNA Artificial Sequence Min3 oligo 112 tcgctaggca ggaaagcggcagccttcagg gtccaggcag ccacgaactt ggccttatac 60 aggattccgg 70 113 71 DNAArtificial Sequence Min4 oligo 113 ctttcctgcc tagcgatttc tttcctagcgtgaagctgac cccactgtgc gtgaccctgt 60 atatggatga c 71 114 70 DNAArtificial Sequence Min5 oligo 114 cgtacctgga cagtcccagc ttctcgaagttgatgatgct ggctcccagc accacgtcat 60 ccatatacag 70 115 71 DNA ArtificialSequence Min6 oligo 115 ggactgtcca gatacgtggc taggctgatc ctgaaggagcctgtgcacgg cgtgtccacc 60 ctgccagaga c 71 116 70 DNA Artificial SequenceMin7 oligo 116 gctcagccac ttccacacag gcactccata gtacacggtc ctcctcaccacggtggtctc 60 tggcagggtg 70 117 51 DNA Artificial Sequence Min8 oligo117 gtggaagtgg ctgagcctgc tggtgccctt tgtgggtacc tgatctagag c 51 118 20DNA Artificial Sequence flanking primer 5′ 118 gcgcaagaag cttgctagcg 20119 21 DNA Artificial Sequence flanking primer 3′ 119 gctctagatcaggtacccca c 21 120 30 DNA Artificial Sequence primer Min-ovaR 120tggacagtcc cactcccagc accacgtcat 30 121 30 DNA Artificial Sequenceprimer Min-ovaF 121 gctgggagtg ggactgtcca ggtacgtggc 30 122 29 DNAArtificial Sequence primer Min-StopR 122 ggtacctcac acaaagggca ccagcaggc29 123 8 PRT Artificial Sequence HBV Env-HIV Pol 476 123 Leu Leu Val ProPhe Val Ile Leu 1 5 124 14 PRT Artificial Sequence HBVcore128 124 ThrPro Pro Ala Tyr Arg Pro Pro Asn Ala Pro Ile Leu Phe 1 5 10 125 9 PRTArtificial Sequence HBV Pol 551-V (peptide 1090.77) 125 Tyr Met Asp AspVal Val Leu Gly Val 1 5 126 30 DNA Artificial Sequence first reactionamplification primer for pMin.1-No PADRE 126 atcgctaggc aggaacttatacaggattcc 30 127 27 DNA Artificial Sequence first reactionamplification primer for pMin.1-Anchor 127 tggacagtcc ggctcccagc accacgt27 128 18 DNA Artificial Sequence 3′ amplification primer (No PADRE) 128ttcctgccta gcgatttc 18 129 27 DNA Artificial Sequence 3′ amplificationprimer (Anchor) 129 gctgggagcc ggactgtcca ggtacgt 27 130 42 DNAArtificial Sequence PCR amplification primer for Ig signal sequencedeletion from pMin.1 for pMin.1-No Sig 130 gctagcgccg ccaccatgcacaccctgtgg aaggccggaa tc 42 131 39 DNA Artificial Sequence pMin.1-Switch5′ fragment amplification primer 131 gggcaccagc aggctcagcc acactcccagcaccacgtc 39 132 40 DNA Artificial Sequence pMin.1-Switch secondoverlapping fragment amplification primer 132 agcctgctgg tgccctttgtgatcctgaag gagcctgtgc 40 133 41 DNA Artificial Sequence pMin.1-Switchsecond overlapping fragment amplification primer 133 agccacgtacctggacagtc ccttccacac aggcactcca t 41 134 32 DNA Artificial SequencepMin.1-Switch 3′ third fragment amplification primer 134 tgtccaggtacgtggctagg ctgtgaggta cc 32 135 42 DNA Artificial Sequence PCRamplification primer Min.0-No Sig-5′ for deletion of signal sequencefrom pMin.0 for pMin.2-GFP 135 gctagcgccg ccaccatgca caccctgtggaaggccggaa tc 42 136 10 PRT Artificial Sequence HBV Env 335-HBV Pol 551136 Val Leu Gly Val Trp Leu Ser Leu Leu Val 1 5 10 137 15 PRT ArtificialSequence HBV POL 661 (peptide 1298.06) 137 Lys Gln Ala Phe Thr Phe SerPro Thr Tyr Lys Ala Phe Leu Cys 1 5 10 15 138 15 PRT Artificial SequenceHBV POL 412 (peptide F107.03) 138 Leu Gln Ser Leu Thr Asn Leu Leu SerSer Asn Leu Ser Trp Leu 1 5 10 15 139 15 PRT Artificial Sequence HBV ENV180 (peptide 1280.06) 139 Ala Gly Phe Phe Leu Leu Thr Arg Ile Leu ThrIle Pro Gln Ser 1 5 10 15 140 15 PRT Artificial Sequence HBV POL 774(peptide 1280.09) 140 Gly Thr Ser Phe Val Tyr Val Pro Ser Ala Leu AsnPro Ala Asp 1 5 10 15 141 20 PRT Artificial Sequence HBV NUC 120(peptide CF-08) 141 Val Ser Phe Gly Val Trp Ile Arg Thr Pro Pro Ala TyrArg Pro Pro 1 5 10 15 Asn Ala Pro Ile 20 142 15 PRT Artificial SequenceHBV NUC 123 (peptide 27.0280) 142 Gly Val Trp Ile Arg Thr Pro Pro AlaTyr Arg Pro Pro Asn Ala 1 5 10 15 143 15 PRT Artificial Sequence HBV NUC121 (peptide 1186.25) 143 Ser Phe Gly Val Trp Ile Arg Thr Pro Pro AlaTyr Arg Pro Pro 1 5 10 15 144 15 PRT Artificial Sequence HBV POL 145(peptide 27.0281) 144 Arg His Tyr Leu His Thr Leu Trp Lys Ala Gly IleLeu Tyr Lys 1 5 10 15 145 15 PRT Artificial Sequence HBV POL 523(peptide F107.04) 145 Pro Phe Leu Leu Ala Gln Phe Thr Ser Ala Ile CysSer Val Val 1 5 10 15 146 15 PRT Artificial Sequence HBV ENV 339(peptide 1186.15) 146 Leu Val Pro Phe Val Gln Trp Phe Val Gly Leu SerPro Thr Val 1 5 10 15 147 15 PRT Artificial Sequence HBV POL 501(peptide 1280.15) 147 Leu His Leu Tyr Ser His Pro Ile Ile Leu Gly PheArg Lys Ile 1 5 10 15 148 15 PRT Artificial Sequence HBV POL 615(peptide 1298.04) 148 Lys Gln Cys Phe Arg Lys Leu Pro Val Asn Arg ProIle Asp Trp 1 5 10 15 149 15 PRT Artificial Sequence HBV POL 764(peptide 1298.07) 149 Ala Ala Asn Trp Ile Leu Arg Gly Thr Ser Phe ValTyr Val Pro 1 5 10 15 150 20 PRT Artificial Sequence HBV CORE 50(peptide 857.02) 150 Pro His His Thr Ala Leu Arg Gln Ala Ile Leu Cys TrpGly Glu Leu 1 5 10 15 Met Thr Leu Ala 20 151 15 PRT Artificial SequenceHBV POL 683 (peptide 35.0100) 151 Leu Cys Gln Val Phe Ala Asp Ala ThrPro Thr Gly Trp Gly Leu 1 5 10 15 152 15 PRT Artificial Sequence HBV POL387 (peptide 35.0096) 152 Glu Ser Arg Leu Val Val Asp Phe Ser Gln PheSer Arg Gly Asn 1 5 10 15 153 15 PRT Artificial Sequence HBV POL 96(peptide 35.0093) 153 Val Gly Pro Leu Thr Val Asn Glu Lys Arg Arg LeuLys Leu Ile 1 5 10 15 154 15 PRT Artificial Sequence HBV POL 422(peptide 1186.18) 154 Asn Leu Ser Trp Leu Ser Leu Asp Val Ser Ala AlaPhe Tyr His 1 5 10 15 155 9 PRT Artificial Sequence HBV ENV ayw 183(peptide 777.03) 155 Phe Leu Leu Thr Arg Ile Leu Thr Ile 1 5 156 9 PRTArtificial Sequence HBV ayw pol 642 (peptide 927.15) 156 Ala Leu Met ProLeu Tyr Ala Cys Ile 1 5 157 9 PRT Artificial Sequence HBV pol 562(peptide 927.11) 157 Phe Leu Leu Ser Leu Gly Ile His Leu 1 5 158 9 PRTArtificial Sequence HBV POL 531 (peptide 1090.11) 158 Ser Ala Ile CysSer Val Val Arg Arg 1 5 159 10 PRT Artificial Sequence HBV pol 665(peptide 1090.10) 159 Gln Ala Phe Thr Phe Ser Pro Thr Tyr Lys 1 5 10 1609 PRT Artificial Sequence HBV pol 47 (peptide 1069.16) 160 Asn Val SerIle Pro Trp Thr His Lys 1 5 161 10 PRT Artificial Sequence HBV pol 388(peptide 1069.20) 161 Leu Val Val Asp Phe Ser Gln Phe Ser Arg 1 5 10 1629 PRT Artificial Sequence HBV adr POL 629 (peptide 1142.05) (peptide1.0166) 162 Lys Val Gly Asn Phe Thr Gly Leu Tyr 1 5 163 10 PRTArtificial Sequence HBV pol 150 (peptide 1069.15) 163 Thr Leu Trp LysAla Gly Ile Leu Tyr Lys 1 5 10 164 9 PRT Artificial Sequence HBV ENV 313(peptide 1145.04) 164 Ile Pro Ile Pro Ser Ser Trp Ala Phe 1 5 165 9 PRTArtificial Sequence HBV core 19-27 (peptide 988.05) 165 Leu Pro Ser AspPhe Phe Pro Ser Val 1 5 166 10 PRT Artificial Sequence HBV POL 354(peptide 1147.04) 166 Thr Pro Ala Arg Val Thr Gly Gly Val Phe 1 5 10 16710 PRT Artificial Sequence HBV env 338-347 (peptide 1069.06) 167 Leu LeuVal Pro Phe Val Gln Trp Phe Val 1 5 10 168 10 PRT Artificial SequenceHBV POL 513 (peptide 1147.13) 168 Phe Leu Leu Ala Gln Phe Thr Ser AlaIle 1 5 10 169 11 PRT Artificial Sequence HBV ENV 259 (peptide 1147.14)169 Val Leu Leu Asp Tyr Gln Gly Met Leu Pro Val 1 5 10 170 9 PRTArtificial Sequence HBV ENV 339 (peptide 1132.01) 170 Leu Val Pro PheVal Gln Trp Phe Val 1 5 171 9 PRT Artificial Sequence HBV pol 504-512(peptide1069.05) 171 Leu Leu Ala Gln Phe Thr Ser Ala Ile 1 5 172 9 PRTArtificial Sequence HBV pol 411 (peptide 927.42) 172 Asn Leu Ser Trp LeuSer Leu Asp Val 1 5 173 9 PRT Artificial Sequence HBV pol 992 (peptide927.41) 173 Leu Leu Ser Ser Asn Leu Ser Trp Leu 1 5 174 9 PRT ArtificialSequence HBV pol 489 (peptide 927.46) 174 Lys Leu His Leu Tyr Ser HisPro Ile 1 5 175 9 PRT Artificial Sequence HBV pol 503 (peptide 1069.071)175 Phe Leu Leu Ala Gln Phe Thr Ser Ala 1 5 176 9 PRT ArtificialSequence HBV ENV 62 (peptide 1142.07) 176 Gly Leu Leu Gly Trp Ser ProGln Ala 1 5 177 9 PRT Artificial Sequence HBV ayw pol 1076 (peptide927.47) 177 His Leu Tyr Ser His Pro Ile Ile Leu 1 5 178 9 PRT ArtificialSequence HBV env 377-385 (peptide 1069.13) 178 Pro Leu Leu Pro Ile PhePhe Cys Leu 1 5 179 9 PRT Artificial Sequence HBV adr-ENV 177 (peptide1013.1402) 179 Val Leu Gln Ala Gly Phe Phe Leu Leu 1 5 180 11 PRTArtificial Sequence HBV pol 376 (peptide 26.0539) 180 Arg Leu Val ValAsp Phe Ser Gln Phe Ser Arg 1 5 10 181 11 PRT Artificial Sequence HBV Xnuc fus 299 (peptide 26.0535) 181 Gly Val Trp Ile Arg Thr Pro Pro AlaTyr Arg 1 5 10 182 9 PRT Artificial Sequence HBV X 64 (peptide 26.0153)182 Ser Ser Ala Gly Pro Cys Ala Leu Arg 1 5 183 9 PRT ArtificialSequence HBV adr “X” 1548 (peptide 1.0993) 183 Lys Val Phe Val Leu GlyGly Cys Arg 1 5 184 9 PRT Artificial Sequence HBV X 69 (peptide 26.0149)184 Cys Ala Leu Arg Phe Thr Ser Ala Arg 1 5 185 8 PRT ArtificialSequence HBV x nuc fus 296 (peptide 26.0023) 185 Val Ser Phe Gly Val TrpIle Arg 1 5 186 11 PRT Artificial Sequence HBV x nuc fus 318 (peptide26.0545) 186 Thr Leu Pro Glu Thr Thr Val Val Arg Arg Arg 1 5 10 187 9PRT Artificial Sequence HBV POL 524 (peptide 20.0131) 187 Ser Val ValArg Arg Ala Phe Pro His 1 5 188 9 PRT Artificial Sequence HBV adr “X”1550 (peptide 1.0219) 188 Phe Val Leu Gly Gly Cys Arg His Lys 1 5 189 8PRT Artificial Sequence HBV pol 656 (peptide 26.0008) 189 Phe Thr PheSer Pro Thr Tyr Lys 1 5 190 9 PRT Artificial Sequence HBV POL 655(peptide 20.0130) 190 Ala Phe Thr Glu Ser Pro Thr Tyr Lys 1 5 191 10 PRTArtificial Sequence HBV POL 530 (peptide 1147.05) 191 Phe Pro His CysLeu Ala Phe Ser Tyr Met 1 5 10 192 9 PRT Artificial Sequence HBV POL 640(peptide 1147.08) 192 Tyr Pro Ala Leu Met Pro Leu Tyr Ala 1 5 193 9 PRTArtificial Sequence HBV X 58 (peptide 1147.06) 193 Leu Pro Val Cys AlaPhe Ser Ser Ala 1 5 194 9 PRT Artificial Sequence HBV POL 429 (peptide1147.02) 194 His Pro Ala Ala Met Pro His Leu Leu 1 5 195 11 PRTArtificial Sequence HBV pol 640 (peptide 26.0570) 195 Tyr Pro Ala LeuMet Pro Leu Tyr Ala Cys Ile 1 5 10 196 8 PRT Artificial Sequence HBV POL640 (peptide 19.0014) 196 Tyr Pro Ala Leu Met Pro Leu Tyr 1 5 197 9 PRTArtificial Sequence HBV POL 541 (peptide 1145.08) 197 Phe Pro His CysLeu Ala Phe Ser Tyr 1 5 198 9 PRT Artificial Sequence HBV NUC 131(peptide 1090.02) 198 Ala Tyr Arg Pro Pro Asn Ala Pro Ile 1 5 199 10 PRTArtificial Sequence HBV adr CORE 419 (peptide 1.0519) 199 Asp Leu LeuAsp Thr Ala Ser Ala Leu Tyr 1 5 10 200 10 PRT Artificial Sequence HBVNUC 117 (peptide 13.0129) 200 Glu Tyr Leu Val Ser Phe Gly Val Trp Ile 15 10 201 10 PRT Artificial Sequence HBV POL 631 (peptide 20.0254) 201Phe Ala Ala Pro Phe Thr Gln Cys Gly Tyr 1 5 10 202 9 PRT ArtificialSequence HBV ALL 1224 (peptide 2.0060) 202 Gly Tyr Pro Ala Leu Met ProLeu Tyr 1 5 203 10 PRT Artificial Sequence HBV pol 149 (peptide 1069.04)203 His Thr Leu Trp Lys Ala Gly Ile Leu Tyr 1 5 10 204 10 PRT ArtificialSequence HBV env 249-258 (peptide 1069.08) 204 Ile Leu Leu Leu Cys LeuIle Phe Leu Leu 1 5 10 205 9 PRT Artificial Sequence HBV POL 745(peptide 1069.23) 205 Lys Tyr Thr Ser Phe Pro Trp Leu Leu 1 5 206 9 PRTArtificial Sequence HBV core 59 (peptide 1069.01) 206 Leu Leu Asp ThrAla Ser Ala Leu Tyr 1 5 207 10 PRT Artificial Sequence HBV ALL 1000(peptide 2.0239) 207 Leu Ser Leu Asp Val Ser Ala Ala Phe Tyr 1 5 10 20810 PRT Artificial Sequence HBV POL 492 (peptide 2.0181) 208 Leu Tyr SerHis Pro Ile Ile Leu Gly Phe 1 5 10 209 10 PRT Artificial Sequence HBV360 (peptide 1039.01) 209 Met Met Trp Tyr Trp Gly Pro Ser Leu Tyr 1 5 10210 9 PRT Artificial Sequence HBV adr 1521 (peptide 2.0126) 210 Met SerThr Thr Asp Leu Glu Ala Tyr 1 5 211 10 PRT Artificial Sequence HBV pol124 (peptide 1069.03) 211 Pro Leu Asp Lys Gly Ile Lys Pro Tyr Tyr 1 5 10212 9 PRT Artificial Sequence HBV pol 808 (peptide 1090.09) 212 Pro ThrThr Gly Arg Thr Ser Leu Tyr 1 5 213 9 PRT Artificial Sequence HBV POL 51(peptide 20.0138) 213 Pro Trp Thr His Lys Val Gly Asn Phe 1 5 214 9 PRTArtificial Sequence HBV ENV 236 (peptide 20.0135) 214 Arg Trp Met CysLeu Arg Arg Phe Ile 1 5 215 10 PRT Artificial Sequence HBV ENV 236(peptide 20.0269) 215 Arg Trp Met Cys Leu Arg Arg Phe Ile Ile 1 5 10 2169 PRT Artificial Sequence HBV POL 167 (peptide 20.0139) 216 Ser Phe CysGly Ser Pro Tyr Ser Trp 1 5 217 9 PRT Artificial Sequence HBV pol 427(peptide 1069.02) 217 Ser Leu Asp Val Ser Ala Ala Phe Tyr 1 5 218 9 PRTArtificial Sequence HBV ENV 334 (peptide 20.0136) 218 Ser Trp Leu SerLeu Leu Val Pro Phe 1 5 219 10 PRT Artificial Sequence HBV POL 392(peptide 20.0271) 219 Ser Trp Pro Lys Phe Ala Val Pro Asn Leu 1 5 10 2209 PRT Artificial Sequence HBV ENV 197 (peptide 20.0137) 220 Ser Trp TrpThr Ser Leu Asn Phe Leu 1 5 221 10 PRT Artificial Sequence HBV POL 4(peptide 2.0173) 221 Ser Tyr Gln His Phe Arg Lys Leu Leu Leu 1 5 10 2229 PRT Artificial Sequence HBV NUC 102 (peptide 13.0073) 222 Trp Phe HisIle Ser Cys Leu Thr Phe 1 5 223 10 PRT Artificial Sequence HBV adr CORE416 (peptide 1.0774) 223 Trp Leu Trp Gly Met Asp Ile Asp Pro Tyr 1 5 10224 11 PRT Artificial Sequence HBV env 359 (peptide 1039.06) 224 Trp MetMet Trp Tyr Trp Gly Pro Ser Leu Tyr 1 5 10 225 10 PRT ArtificialSequence HBv 18-27 I10 var. (peptide 924.14) 225 Phe Leu Pro Ser Asp PhePhe Pro Ser Ile 1 5 10 226 10 PRT Artificial Sequence HBc 18-27 analog(peptide 941.01) 226 Phe Leu Pro Ser Asp Tyr Phe Pro Ser Val 1 5 10 22711 PRT Artificial Sequence HBV core 141-151 analog (peptide 1083.02) 227Ser Thr Leu Pro Glu Thr Tyr Val Val Arg Arg 1 5 10 228 9 PRT ArtificialSequence HBV ENV 313 analog (peptide 1145.05) 228 Phe Pro Ile Pro SerSer Trp Ala Phe 1 5 229 9 PRT Artificial Sequence HBV POL 541 analog(peptide 1145.11) 229 Phe Pro His Cys Leu Ala Phe Ser Leu 1 5 230 9 PRTArtificial Sequence HBV POL 541 analog (peptide 1145.24) 230 Phe Pro HisCys Leu Ala Phe Ala Leu 1 5 231 9 PRT Artificial Sequence HBV ENV 313analog (peptide 1145.06) 231 Ile Pro Ile Thr Ser Ser Trp Ala Phe 1 5 2329 PRT Artificial Sequence HBV ENV 313 analog (peptide 1145.23) 232 IlePro Ile Pro Met Ser Trp Ala Phe 1 5 233 9 PRT Artificial Sequence HBVENV 313 analog (peptide 1145.07) 233 Ile Pro Ile Leu Ser Ser Trp Ala Phe1 5 234 9 PRT Artificial Sequence HBV POL 541 analog (peptide 1145.09)234 Phe Pro Val Cys Leu Ala Phe Ser Tyr 1 5 235 9 PRT ArtificialSequence HBV POL 541 analog (peptide 1145.10) 235 Phe Pro His Cys LeuAla Phe Ala Tyr 1 5 236 26 PRT Artificial Sequence HCV NS3 1242-1267 236Ala Ala Tyr Ala Ala Gln Gly Tyr Lys Val Leu Val Leu Asn Pro Ser 1 5 1015 Val Ala Ala Thr Leu Gly Phe Gly Ala Tyr 20 25 237 20 PRT ArtificialSequence HCV NS3 1242 (peptide P98.03) 237 Ala Ala Tyr Ala Ala Gln GlyTyr Lys Val Leu Val Leu Asn Pro Ser 1 5 10 15 Val Ala Ala Thr 20 238 20PRT Artificial Sequence HCV NS3 1248 (peptide P98.04) 238 Gly Tyr LysVal Leu Val Leu Asn Pro Ser Val Ala Ala Thr Leu Gly 1 5 10 15 Phe GlyAla Tyr 20 239 14 PRT Artificial Sequence HCV NS3 1248 (peptide P98.05)239 Gly Tyr Lys Val Leu Val Leu Asn Pro Ser Val Ala Ala Thr 1 5 10 24015 PRT Artificial Sequence HCV NS3 1253 (peptide 1283.21) 240 Gly TyrLys Val Leu Val Leu Asn Pro Ser Val Ala Ala Thr Leu 1 5 10 15 241 15 PRTArtificial Sequence HCV NS3 1251 (peptide 1283.20) 241 Ala Gln Gly TyrLys Val Leu Val Leu Asn Pro Ser Val Ala Ala 1 5 10 15 242 22 PRTArtificial Sequence HCV NS4 1914-1935 242 Gly Glu Gly Ala Val Gln TrpMet Asn Arg Leu Ile Ala Phe Ala Ser 1 5 10 15 Arg Gly Asn His Val Ser 20243 21 PRT Artificial Sequence HCV NS4 1914 (peptide F134.08) 243 GlyGlu Gly Ala Val Gln Trp Met Asn Arg Leu Ile Ala Phe Ala Ser 1 5 10 15Arg Gly Asn His Val 20 244 15 PRT Artificial Sequence HCV NS4 1921(peptide 1283.44) 244 Met Asn Arg Leu Ile Ala Phe Ala Ser Arg Gly AsnHis Val Ser 1 5 10 15 245 15 PRT Artificial Sequence HCV NS3 1025(peptide 1283.16) 245 Ser Lys Gly Trp Arg Leu Leu Ala Pro Ile Thr AlaTyr Ala Gln 1 5 10 15 246 15 PRT Artificial Sequence HCV NS5 2641(peptide 1283.55) 246 Gly Ser Ser Tyr Gly Phe Gln Tyr Ser Pro Gly GlnArg Val Glu 1 5 10 15 247 20 PRT Artificial Sequence HCV NS4 1772(peptide F134.05) 247 Asn Phe Ile Ser Gly Ile Gln Tyr Leu Ala Gly LeuSer Thr Leu Pro 1 5 10 15 Gly Asn Pro Ala 20 248 15 PRT ArtificialSequence HCV NS5 2939 (peptide 1283.61) 248 Ala Ser Cys Leu Arg Lys LeuGly Val Pro Pro Leu Arg Val Trp 1 5 10 15 249 15 PRT Artificial SequenceHCV NS3 1393 (peptide 1283.25) 249 Gly Arg His Leu Ile Phe Cys His SerLys Lys Lys Cys Asp Glu 1 5 10 15 250 15 PRT Artificial Sequence HCV1466 (peptide 35.0107) 250 Thr Val Asp Phe Ser Leu Asp Pro Thr Phe ThrIle Glu Thr Thr 1 5 10 15 251 15 PRT Artificial Sequence HCV 1437(peptide 35.0106) 251 Val Val Val Val Ala Thr Asp Ala Leu Met Thr GlyTyr Thr Gly 1 5 10 15 252 9 PRT Artificial Sequence HCV NS1/E2 728(peptide 1090.18) 252 Phe Leu Leu Leu Ala Asp Ala Arg Val 1 5 253 10 PRTArtificial Sequence HCV NS4 1812 (peptide 1073.05) 253 Leu Leu Phe AsnIle Leu Gly Gly Trp Val 1 5 10 254 9 PRT Artificial Sequence HCV NS31590 (peptide 1013.02) 254 Tyr Leu Val Ala Tyr Gln Ala Thr Val 1 5 255 9PRT Artificial Sequence HCV Core 132 (peptide 1013.1002) 255 Asp Leu MetGly Tyr Ile Pro Leu Val 1 5 256 10 PRT Artificial Sequence HCV NS5 2611(peptide 1090.22) 256 Arg Leu Ile Val Phe Pro Asp Leu Gly Val 1 5 10 2579 PRT Artificial Sequence HCV NS4 1666 (peptide 24.0075) 257 Val Leu ValGly Gly Val Leu Ala Ala 1 5 258 9 PRT Artificial Sequence HCV NS4 1920(peptide 24.0073) 258 Trp Met Asn Arg Leu Ile Ala Phe Ala 1 5 259 9 PRTArtificial Sequence HCV NS4 1769 (peptide 1174.08) 259 His Met Trp AsnPhe Ile Ser Gly Ile 1 5 260 9 PRT Artificial Sequence HCV NS4 1851(peptide 1073.06) 260 Ile Leu Ala Gly Tyr Gly Ala Gly Val 1 5 261 9 PRTArtificial Sequence HCV NS1/E2 726 (peptide 24.0071) 261 Leu Leu Phe LeuLeu Leu Ala Asp Ala 1 5 262 10 PRT Artificial Sequence HCV Core 35(peptide 1073.07) 262 Tyr Leu Leu Pro Arg Arg Gly Pro Arg Leu 1 5 10 2639 PRT Artificial Sequence HCV NS3 1136 (peptide 1.0119) 263 Tyr Leu ValThr Arg His Ala Asp Val 1 5 264 9 PRT Artificial Sequence HCV Core 51(peptide 1.0952) 264 Lys Thr Ser Glu Arg Ser Gln Pro Arg 1 5 265 10 PRTArtificial Sequence HCV NS4 1863 (peptide 1073.10) 265 Gly Val Ala GlyAla Leu Val Ala Phe Lys 1 5 10 266 9 PRT Artificial Sequence HCV NS31391 (peptide 1.0123) 266 Leu Ile Phe Cys His Ser Lys Lys Lys 1 5 267 9PRT Artificial Sequence HCV E1 290 (peptide 1.0955) 267 Gln Leu Phe ThrPhe Ser Pro Arg Arg 1 5 268 9 PRT Artificial Sequence HCV Core 43(peptide 1073.11) 268 Arg Leu Gly Val Arg Ala Thr Arg Lys 1 5 269 10 PRTArtificial Sequence HCV NS1/E2 635 (peptide 1073.13) 269 Arg Met Tyr ValGly Gly Val Glu His Arg 1 5 10 270 9 PRT Artificial Sequence HCV NS41864 (peptide 24.0090) 270 Val Ala Gly Ala Leu Val Ala Phe Lys 1 5 271 9PRT Artificial Sequence HCV NS5 3036 (peptide F104.01) 271 Val Gly IleTyr Leu Leu Pro Asn Arg 1 5 272 9 PRT Artificial Sequence HCV Core 168(peptide 1145.12) 272 Leu Pro Gly Cys Ser Phe Ser Ile Phe 1 5 273 8 PRTArtificial Sequence HCV 1378 (peptide 29.0035) 273 Ile Pro Phe Tyr GlyLys Ala Ile 1 5 274 9 PRT Artificial Sequence HCV NS3 1128 (peptide1069.62) 274 Cys Thr Cys Gly Ser Ser Asp Leu Tyr 1 5 275 9 PRTArtificial Sequence HCV NS4 1765 (peptide 24.0092) 275 Phe Trp Ala LysHis Met Trp Asn Phe 1 5 276 9 PRT Artificial Sequence HCV NS5 2922(peptide 13.0019) 276 Leu Ser Ala Phe Ser Leu His Ser Tyr 1 5 277 9 PRTArtificial Sequence HCV NS3 1267 (peptide 24.0086) 277 Leu Gly Phe GlyAla Tyr Met Ser Lys 1 5 278 9 PRT Artificial Sequence HCV NS5 2621(peptide 1174.21) 278 Arg Val Cys Glu Lys Met Ala Leu Tyr 1 5 279 9 PRTArtificial Sequence HCV NS1/E2 557 (peptide 1174.16) 279 Trp Met Asn SerThr Gly Phe Thr Lys 1 5 280 10 PRT Artificial Sequence HCV NS3 1622(peptide 1073.04) 280 Thr Leu His Gly Pro Thr Pro Leu Leu Tyr 1 5 10 2819 PRT Artificial Sequence HCV NS3 1588 (peptide 16.0012) 281 Phe Pro TyrLeu Val Ala Tyr Gln Ala 1 5 282 9 PRT Artificial Sequence HCV NS1/E2 623(peptide 15.0047) 282 Tyr Pro Cys Thr Val Asn Phe Thr Ile 1 5 283 10 PRTArtificial Sequence HCV NS5 2129 (peptide 24.0093) 283 Glu Val Asp GlyVal Arg Leu His Arg Tyr 1 5 10 284 11 PRT Artificial Sequence HCV 126(peptide 3.0417) 284 Leu Thr Cys Gly Phe Ala Asp Leu Met Gly Tyr 1 5 10285 9 PRT Artificial Sequence HCV E1 700 (peptide 1073.01) 285 Asn IleVal Asp Val Gln Tyr Leu Tyr 1 5 286 10 PRT Artificial Sequence HCV NS52921 (peptide 1.0509) 286 Gly Leu Ser Ala Phe Ser Leu His Ser Tyr 1 5 10287 11 PRT Artificial Sequence HCV E1 275 (peptide 1073.17) 287 Met TyrVal Gly Asp Leu Cys Gly Ser Val Phe 1 5 10 288 10 PRT ArtificialSequence HCV NS1/E2 633 (peptide 1073.18) 288 Met Tyr Val Gly Gly ValGlu His Arg Leu 1 5 10 289 9 PRT Artificial Sequence HCV NS4 1778(peptide 13.075) 289 Gln Tyr Leu Ala Gly Leu Ser Thr Leu 1 5 290 9 PRTArtificial Sequence HCV Core 168 (peptide 1145.13) 290 Phe Pro Gly CysSer Phe Ser Ile Phe 1 5 291 9 PRT Artificial Sequence HCV Core 168(peptide 1145.25) 291 Leu Pro Gly Cys Met Phe Ser Ile Phe 1 5 292 9 PRTArtificial Sequence HCV Core 169 (peptide 1292.24) 292 Leu Pro Gly CysSer Phe Ser Ile Ile 1 5 293 9 PRT Artificial Sequence HCV Core 168(peptide 1145.14) 293 Leu Pro Val Cys Ser Phe Ser Ile Phe 1 5 294 9 PRTArtificial Sequence HCV Core 168 (peptide 1145.15) 294 Leu Pro Gly CysSer Phe Ser Tyr Phe 1 5 295 26 PRT Artificial Sequence HIV1 GAG 294-319295 Gly Glu Ile Tyr Lys Arg Trp Ile Ile Leu Gly Leu Asn Lys Ile Val 1 510 15 Arg Met Tyr Ser Pro Thr Ser Ile Leu Asp 20 25 296 22 PRTArtificial Sequence HIV gag 298-319 296 Lys Arg Trp Ile Ile Leu Gly LeuAsn Lys Ile Val Arg Met Tyr Ser 1 5 10 15 Pro Thr Ser Ile Leu Asp 20 29715 PRT Artificial Sequence HIV1 GAG 298 (peptide 27.0313) 297 Lys ArgTrp Ile Ile Leu Gly Leu Asn Lys Ile Val Arg Met Tyr 1 5 10 15 298 15 PRTArtificial Sequence HIV1 GAG 294 (peptide 27.0311) 298 Gly Glu Ile TyrLys Arg Trp Ile Ile Leu Gly Leu Asn Lys Ile 1 5 10 15 299 15 PRTArtificial Sequence HIV1 POL 596 (peptide 27.0354) 299 Trp Glu Phe ValAsn Thr Pro Pro Leu Val Lys Leu Trp Tyr Gln 1 5 10 15 300 15 PRTArtificial Sequence HIV1 POL 956 (peptide 27.0377) 300 Gln Lys Gln IleThr Lys Ile Gln Asn Phe Arg Val Tyr Tyr Arg 1 5 10 15 301 16 PRTArtificial Sequence HIV1 POL 711-726 301 Glu Lys Val Tyr Leu Ala Trp ValPro Ala His Lys Gly Ile Gly Gly 1 5 10 15 302 15 PRT Artificial SequenceHIV POL 712 (peptide 1280.03) 302 Lys Val Tyr Leu Ala Trp Val Pro AlaHis Lys Gly Ile Gly Gly 1 5 10 15 303 15 PRT Artificial Sequence HIV1POL 711 (peptide 27.0361) 303 Glu Lys Val Tyr Leu Ala Trp Val Pro AlaHis Lys Gly Ile Gly 1 5 10 15 304 22 PRT Artificial Sequence HIV1 gag165-186 304 Pro Ile Val Gln Asn Ile Gln Gly Gln Met Val His Gln Ala IleSer 1 5 10 15 Pro Arg Thr Leu Asn Ala 20 305 15 PRT Artificial SequenceHIV1 GAG 171 (peptide 27.0304) 305 Gln Gly Gln Met Val His Gln Ala IleSer Pro Arg Thr Leu Asn 1 5 10 15 306 15 PRT Artificial Sequence HIV1ENV 729 (peptide 27.0297) 306 Gln His Leu Leu Gln Leu Thr Val Trp GlyIle Lys Gln Leu Gln 1 5 10 15 307 15 PRT Artificial Sequence HIV1 POL335 (peptide 27.0344) 307 Ser Pro Ala Ile Phe Gln Ser Ser Met Thr LysIle Leu Glu Pro 1 5 10 15 308 16 PRT Artificial Sequence HIV1 ENV 566(peptide F091.15) 308 Ile Lys Gln Phe Ile Asn Met Trp Gln Glu Val GlyLys Ala Met Tyr 1 5 10 15 309 15 PRT Artificial Sequence HIV1 POL 303(peptide 27.0341) 309 Phe Arg Lys Tyr Thr Ala Phe Thr Ile Pro Ser IleAsn Asn Glu 1 5 10 15 310 15 PRT Artificial Sequence HIV1 POL 758(peptide 27.0364) 310 His Ser Asn Trp Arg Ala Met Ala Ser Asp Phe AsnLeu Pro Pro 1 5 10 15 311 15 PRT Artificial Sequence HIV1 POL 915(peptide 27.0373) 311 Lys Thr Ala Val Gln Met Ala Val Phe Ile His AsnPhe Lys Arg 1 5 10 15 312 22 PRT Artificial Sequence HIV GAG 245 312 AspArg Val His Pro Val His Ala Gly Pro Ile Ala Pro Gly Gln Met 1 5 10 15Arg Glu Pro Arg Gly Ser 20 313 26 PRT Artificial Sequence HIV gag195-220 313 Ala Phe Ser Pro Glu Val Ile Pro Met Phe Ser Ala Leu Ser GluGly 1 5 10 15 Ala Thr Pro Gln Asp Leu Asn Thr Met Leu 20 25 314 22 PRTArtificial Sequence HIV gag 195-216 314 Ala Phe Ser Pro Glu Val Ile ProMet Phe Ser Ala Leu Ser Glu Gly 1 5 10 15 Ala Thr Pro Gln Asp Leu 20 31516 PRT Artificial Sequence HIV gag 205 (peptide 200.06) 315 Ser Ala LeuSer Glu Gly Ala Thr Pro Gln Asp Leu Asn Thr Met Leu 1 5 10 15 316 15 PRTArtificial Sequence HIV gag 197 (peptide 27.0307) 316 Ser Pro Glu ValIle Pro Met Phe Ser Ala Leu Ser Glu Gly Ala 1 5 10 15 317 22 PRTArtificial Sequence HIV gag 275 317 Leu Gln Glu Gln Ile Gly Trp Met ThrAsn Asn Pro Pro Ile Pro Val 1 5 10 15 Gly Glu Ile Tyr Lys Arg 20 318 15PRT Artificial Sequence HIV gag 276 (peptide 27.0310) 318 Gln Glu GlnIle Gly Trp Met Thr Asn Asn Pro Pro Ile Pro Val 1 5 10 15 319 15 PRTArtificial Sequence HIV VPU 31 (peptide 35.0135) 319 Tyr Arg Lys Ile LeuArg Gln Arg Lys Ile Asp Arg Leu Ile Asp 1 5 10 15 320 15 PRT ArtificialSequence HIV POL 874 (peptide 35.0131) 320 Trp Ala Gly Ile Lys Gln GluPhe Gly Ile Pro Tyr Asn Pro Gln 1 5 10 15 321 15 PRT Artificial SequenceHIV POL 674 (peptide 35.0127) 321 Glu Val Asn Ile Val Thr Asp Ser GlnTyr Ala Leu Gly Ile Ile 1 5 10 15 322 15 PRT Artificial Sequence HIV POL619 (peptide 35.0125) 322 Ala Glu Thr Phe Tyr Val Asp Gly Ala Ala AsnArg Glu Thr Lys 1 5 10 15 323 15 PRT Artificial Sequence HIV POL 989(peptide 35.0133) 323 Gly Ala Val Val Ile Gln Asp Asn Ser Asp Ile LysVal Val Pro 1 5 10 15 324 10 PRT Artificial Sequence HIV1 POL 70(peptide 25.0148) 324 Met Ala Ser Asp Phe Asn Leu Pro Pro Val 1 5 10 3259 PRT Artificial Sequence HIV gag 397 (peptide 1069.32) 325 Val Leu AlaGlu Ala Met Ser Gln Val 1 5 326 9 PRT Artificial Sequence HIV1 POL 87(peptide 25.0062) 326 Lys Leu Val Gly Lys Leu Asn Trp Ala 1 5 327 9 PRTArtificial Sequence HIV1 NEF 62 (peptide 25.0039) 327 Leu Thr Phe GlyTrp Cys Phe Lys Leu 1 5 328 9 PRT Artificial Sequence HIV1 GAG 34(peptide 25. 0035) 328 Met Thr Asn Asn Pro Pro Ile Pro Val 1 5 329 9 PRTArtificial Sequence HIV1 VPR 72 (peptide 25.0057) 329 Arg Ile Leu GlnGln Leu Leu Phe Ile 1 5 330 9 PRT Artificial Sequence HIV POL 1434(peptide 1.0944) 330 Ala Val Phe Ile His Asn Phe Lys Arg 1 5 331 10 PRTArtificial Sequence HIV POL 1474 (peptide 1.1056) 331 Lys Ile Gln AsnPhe Arg Val Tyr Tyr Arg 1 5 10 332 10 PRT Artificial Sequence HIV pol1432 (peptide 1069.49) 332 Gln Met Ala Val Phe Ile His Asn Phe Lys 1 510 333 9 PRT Artificial Sequence HIV pol 337 (peptide 966.0102) 333 AlaIle Phe Gln Ser Ser Met Thr Lys 1 5 334 9 PRT Artificial Sequence HIVpol 909 (peptide 1150.14) 334 Met Ala Val Phe Ile His Asn Phe Lys 1 5335 10 PRT Artificial Sequence HIV nef 73-82 (peptide 940.03) 335 GlnVal Pro Leu Arg Pro Met Thr Tyr Lys 1 5 10 336 10 PRT ArtificialSequence HIV1 ENV 81 (peptide 25.0175) 336 Thr Thr Leu Phe Cys Ala SerAsp Ala Lys 1 5 10 337 10 PRT Artificial Sequence HIV POL 65 (peptide25.0209) 337 Val Thr Ile Lys Ile Gly Gly Gln Leu Lys 1 5 10 338 9 PRTArtificial Sequence HIV nef 84-92 (peptide 1146.01) 338 Phe Pro Val ArgPro Gln Val Pro Leu 1 5 339 9 PRT Artificial Sequence HIV env 293(peptide 29.0060) 339 Ile Pro Ile His Tyr Cys Ala Pro Ala 1 5 340 9 PRTArtificial Sequence HIV POL 171 (peptide 15.0073) 340 Phe Pro Ile SerPro Ile Glu Thr Val 1 5 341 9 PRT Artificial Sequence HIV env 285(peptide 29.0056) 341 Cys Pro Lys Val Ser Phe Glu Pro Ile 1 5 342 11 PRTArtificial Sequence HIV pol 883 (peptide 29.0107) 342 Ile Pro Tyr AsnPro Gln Ser Gln Gly Val Val 1 5 10 343 10 PRT Artificial Sequence HIV1POL 96 (peptide 25.0151) 343 Cys Thr Leu Asn Phe Pro Ile Ser Pro Ile 1 510 344 10 PRT Artificial Sequence HIV1 NEF 62 (peptide 25.0143) 344 LeuThr Pro Gly Trp Cys Phe Lys Leu Val 1 5 10 345 9 PRT Artificial SequenceHIV1 POL 83 (peptide 25.0043) 345 Tyr Thr Ala Phe Thr Ile Pro Ser Ile 15 346 9 PRT Artificial Sequence HIV1 VPR 76 (peptide 25.0055) 346 AlaIle Ile Arg Ile Leu Gln Gln Leu 1 5 347 9 PRT Artificial Sequence HIV1POL 52 (peptide 25.0049) 347 Ala Leu Val Glu Ile Cys Thr Glu Met 1 5 3489 PRT Artificial Sequence HIV1 ENV 61 (peptide 25.0032) 348 Leu Leu GlnLeu Thr Val Trp Gly Ile 1 5 349 9 PRT Artificial Sequence HIV1 POL 100(peptide 25.0050) 349 Leu Val Gly Pro Thr Pro Val Asn Ile 1 5 350 9 PRTArtificial Sequence HIV1 POL 65 (peptide 25.0047) 350 Lys Ala Ala CysTrp Trp Ala Gly Ile 1 5 351 10 PRT Artificial Sequence HIV1 POL 96(peptide 25.0162) 351 Lys Met Ile Gly Gly Ile Gly Gly Phe Ile 1 5 10 3529 PRT Artificial Sequence HIV1 POL 78 (peptide 25.0052) 352 Arg Ala MetAla Ser Asp Phe Asn Leu 1 5 353 10 PRT Artificial Sequence HIV ENV 814(peptide 1211.09) 353 Ser Leu Leu Asn Ala Thr Asp Ile Ala Val 1 5 10 3549 PRT Artificial Sequence HIV1 POL 96 (peptide 25.0041) 354 Thr Leu AsnPhe Pro Ile Ser Pro Ile 1 5 355 9 PRT Artificial Sequence HIV POL 1075(peptide 1.0046) 355 Ile Val Ile Trp Gly Lys Thr Pro Lys 1 5 356 9 PRTArtificial Sequence HIV1 GAG 45 (peptide 25.0064) 356 Met Val His GlnAla Ile Ser Pro Arg 1 5 357 9 PRT Artificial Sequence HIV POL 1227(peptide 1.0062) 357 Tyr Leu Ala Trp Val Pro Ala His Lys 1 5 358 9 PRTArtificial Sequence HIV POL 859 (peptide 1.0942) 358 Met Thr Lys Ile LeuGlu Pro Phe Arg 1 5 359 10 PRT Artificial Sequence HIV1 GAG 45 (peptide25.0184) 359 Gln Met Val His Gln Ala Ile Ser Pro Arg 1 5 10 360 10 PRTArtificial Sequence HIV pol 1434 (peptide 1069.48) 360 Ala Val Phe IleHis Asn Phe Lys Arg Lys 1 5 10 361 9 PRT Artificial Sequence HIV pol1358 (peptide 1069.44) 361 Lys Leu Ala Gly Arg Trp Pro Val Lys 1 5 36211 PRT Artificial Sequence HIV pol 1225 (peptide 1069.42) 362 Lys ValTyr Leu Ala Trp Val Pro Ala His Lys 1 5 10 363 9 PRT Artificial SequenceHIV pol 752 (peptide 1.0024) 363 Asn Thr Pro Val Phe Ala Ile Lys Lys 1 5364 9 PRT Artificial Sequence HIV1 ENV 53 (peptide 25.0062) 364 Arg IleVal Glu Leu Leu Gly Arg Arg 1 5 365 9 PRT Artificial Sequence HIV1 POL65 (peptide 25.0095) 365 Thr Ile Lys Ile Gly Gly Gln Leu Lys 1 5 366 9PRT Artificial Sequence HIV1 ENV 82 (peptide 25.0078) 366 Thr Leu PheCys Ala Ser Asp Ala Lys 1 5 367 9 PRT Artificial Sequence HIV1 VIF 83(peptide 25.0104) 367 Val Met Ile Val Trp Gln Val Asp Arg 1 5 368 11 PRTArtificial Sequence HIV env 48 (peptide 1069.47) 368 Val Thr Val Tyr TyrGly Val Pro Val Trp Lys 1 5 10 369 10 PRT Artificial Sequence HIV GAG507 (peptide 15.0268) 369 Tyr Pro Leu Ala Ser Leu Arg Ser Leu Phe 1 5 10370 9 PRT Artificial Sequence HIV GAG 248 (peptide 1292.13) 370 His ProVal His Ala Gly Pro Ile Ala 1 5 371 8 PRT Artificial Sequence HIV con.REV 71 (peptide 19.0044) 371 Val Pro Leu Gln Leu Pro Pro Leu 1 5 372 10PRT Artificial Sequence HIV POL 1187 (peptide 1.0431) 372 Glu Val AsnIle Val Thr Asp Ser Gln Tyr 1 5 10 373 9 PRT Artificial Sequence HIV GAG298 (peptide 1.0014) 373 Phe Arg Asp Tyr Val Asp Arg Phe Tyr 1 5 374 9PRT Artificial Sequence HIV1 ENV 69 (peptide 25.0113) 374 Ile Trp GlyCys Ser Gly Lys Leu Ile 1 5 375 9 PRT Artificial Sequence HIV1 VPR 92(peptide 25.0127) 375 Ile Tyr Glu Thr Tyr Gly Asp Thr Trp 1 5 376 9 PRTArtificial Sequence HIV pol 1036 (peptide 1069.60) 376 Ile Tyr Gln GluPro Phe Lys Asn Leu 1 5 377 9 PRT Artificial Sequence HIV pol 359(peptide 2.0129) 377 Ile Tyr Gln Tyr Met Asp Asp Leu Tyr 1 5 378 9 PRTArtificial Sequence HIV1 VPR 56 (peptide 25.0128) 378 Pro Tyr Asn GluTrp Thr Leu Glu Leu 1 5 379 9 PRT Artificial Sequence HIV1 POL 74(peptide 25.0123) 379 Pro Tyr Asn Thr Pro Val Phe Ala Ile 1 5 380 9 PRTArtificial Sequence HIV env 2778 (peptide 1069.57) 380 Arg Tyr Leu LysAsp Gln Gln Leu Leu 1 5 381 9 PRT Artificial Sequence HIV env 2778(peptide 1069.58) 381 Arg Tyr Leu Arg Asp Gln Gln Leu Leu 1 5 382 9 PRTArtificial Sequence HIV pol 1033 (peptide 1069.59) 382 Thr Tyr Gln IleTyr Gln Glu Pro Phe 1 5 383 10 PRT Artificial Sequence HIV pol 358(peptide 1069.27) 383 Val Ile Tyr Gln Tyr Met Asp Asp Leu Tyr 1 5 10 38410 PRT Artificial Sequence HIV POL 265 (peptide 1069.26) 384 Val Thr ValLeu Asp Val Gly Asp Ala Tyr 1 5 10 385 9 PRT Artificial Sequence HIV1ENV 47 (peptide 25.0115) 385 Val Trp Lys Glu Ala Thr Thr Thr Leu 1 5 38610 PRT Artificial Sequence HIV1 ENV 47 (peptide 25.0218) 386 Val Trp LysGlu Ala Thr Thr Thr Leu Phe 1 5 10 387 10 PRT Artificial Sequence HIV1POL 96 (peptide 25.0219) 387 Tyr Met Gln Ala Thr Trp Ile Pro Glu Trp 1 510 388 10 PRT Artificial Sequence HIV MN GP160 814(a) (peptide 1211.4)388 Ser Leu Leu Asn Ala Thr Ala Ile Ala Val 1 5 10 389 9 PRT ArtificialSequence HIV pol 337(a) (peptide F105.21) 389 Ala Ile Phe Gln Arg SerMet Thr Arg 1 5 390 9 PRT Artificial Sequence HIV pol 337(a) (peptideF105.17) 390 Ala Ile Phe Gln Ser Ser Met Thr Arg 1 5 391 9 PRTArtificial Sequence HIV pol 337(a) (peptide F105.02) 391 Gly Ile Phe GlnSer Ser Met Thr Lys 1 5 392 9 PRT Artificial Sequence HIV pol 337(a)(peptide F105.03) 392 Ala Ala Phe Gln Ser Ser Met Thr Lys 1 5 393 9 PRTArtificial Sequence HIV pol 337(a) (peptide F105.04) 393 Ala Ile Ala GlnSer Ser Met Thr Lys 1 5 394 9 PRT Artificial Sequence HIV pol 337(a)(peptide F105.05) 394 Ala Ile Phe Ala Ser Ser Met Thr Lys 1 5 395 9 PRTArtificial Sequence HIV pol 337(a) (peptide F105.06) 395 Ala Ile Phe GlnAla Ser Met Thr Lys 1 5 396 9 PRT Artificial Sequence HIV pol 337(a)(peptide F105.07) 396 Ala Ile Phe Gln Ser Ala Met Thr Lys 1 5 397 9 PRTArtificial Sequence HIV pol 337(a) (peptide F105.08) 397 Ala Ile Phe GlnSer Ser Ala Thr Lys 1 5 398 9 PRT Artificial Sequence HIV pol 337(a)(peptide F105.09) 398 Ala Ile Phe Gln Ser Ser Met Ala Lys 1 5 399 9 PRTArtificial Sequence HIV pol 337(a) (peptide F105.11) 399 Phe Ile Phe GlnSer Ser Met Thr Lys 1 5 400 9 PRT Artificial Sequence HIV pol 337(a)(peptide F105.12) 400 Ser Ile Phe Gln Ser Ser Met Thr Lys 1 5 401 9 PRTArtificial Sequence HIV pol 337(a) (peptide F105.16) 401 Ala Ile Phe GlnCys Ser Met Thr Lys 1 5 402 9 PRT Artificial Sequence HIV nef 84-92analog (peptide 1145.03) 402 Phe Pro Val Arg Pro Gln Phe Pro Leu 1 5 4039 PRT Artificial Sequence HIV nef 84-92(a) (peptide 1181.03) 403 Phe ProVal Arg Pro Gln Val Pro Ile 1 5 404 9 PRT Artificial Sequence HIV GAG248 (peptide 1292.14) 404 His Pro Val His Ala Gly Pro Ile Ile 1 5 405 9PRT Artificial Sequence HIV POL 179 (peptide 1292.09) 405 Phe Pro IleSer Pro Ile Glu Thr Ile 1 5 406 9 PRT Artificial Sequence HIV nef 84-92analog (peptide 1145.02) 406 Phe Pro Val Thr Pro Gln Val Pro Leu 1 5 4079 PRT Artificial Sequence HIV nef 84-92 analog (peptide 1145.22) 407 PhePro Val Arg Met Gln Val Pro Leu 1 5 408 9 PRT Artificial Sequence HIVnef 84-92(a) (peptide 1181.04) 408 Phe Pro Val Arg Pro Gln Val Pro Met 15 409 9 PRT Artificial Sequence HIV nef 84-92(a) (peptide 1181.01) 409Phe Pro Val Arg Pro Gln Val Pro Ala 1 5 410 9 PRT Artificial SequenceHIV nef 84-92(a) (peptide 1181.02) 410 Phe Pro Val Arg Pro Gln Val ProVal 1 5 411 9 PRT Artificial Sequence HIV nef 84-92(a) (peptide 1181.05)411 Phe Pro Val Arg Pro Gln Val Pro Phe 1 5 412 9 PRT ArtificialSequence HIV nef 84-92(a) (peptide 1181.06) 412 Phe Pro Val Arg Pro GlnVal Pro Trp 1 5 413 16 PRT Artificial Sequence Pf SSP2 61 (peptideF125.04) 413 Arg His Asn Trp Val Asn His Ala Val Pro Leu Ala Met Lys LeuIle 1 5 10 15 414 15 PRT Artificial Sequence Pf SSP2 62 (peptide1188.34) 414 His Asn Trp Val Asn His Ala Val Pro Leu Ala Met Lys Leu Ile1 5 10 15 415 15 PRT Artificial Sequence Pf EXP1 71 (peptide1188.16) 415Lys Ser Lys Tyr Lys Leu Ala Thr Ser Val Leu Ala Gly Leu Leu 1 5 10 15416 18 PRT Artificial Sequence Pf LSA1 13 416 Leu Val Asn Leu Leu IlePhe His Ile Asn Gly Lys Ile Ile Lys Asn 1 5 10 15 Ser Glu 417 17 PRTArtificial Sequence Pf LSA 13 (peptide F125.02) 417 Leu Val Asn Leu LeuIle Phe His Ile Asn Gly Lys Ile Ile Lys Asn 1 5 10 15 Ser 418 15 PRTArtificial Sequence Pf LSA1 16 (peptide 27.0402) 418 Leu Leu Ile Phe HisIle Asn Gly Lys Ile Ile Lys Asn Ser Glu 1 5 10 15 419 15 PRT ArtificialSequence Pf SSP2 512 (peptide 1188.32) 419 Gly Leu Ala Tyr Lys Phe ValVal Pro Gly Ala Ala Thr Pro Tyr 1 5 10 15 420 15 PRT Artificial SequencePf CSP 410 (peptide 27.0392) 420 Ser Ser Val Phe Asn Val Val Asn Ser SerIle Gly Leu Ile Met 1 5 10 15 421 15 PRT Artificial Sequence Pf SSP2 223(peptide 27.0417) 421 Val Lys Asn Val Ile Gly Pro Phe Met Lys Ala ValCys Val Glu 1 5 10 15 422 15 PRT Artificial Sequence Pf CSP 2 (peptide27.0388) 422 Met Arg Lys Leu Ala Ile Leu Ser Val Ser Ser Phe Leu Phe Val1 5 10 15 423 15 PRT Artificial Sequence Pf CSP 53 (peptide 27.0387) 423Met Asn Tyr Tyr Gly Lys Gln Glu Asn Trp Tyr Ser Leu Lys Lys 1 5 10 15424 15 PRT Artificial Sequence Pf SSP2 494 (peptide 1188.38) 424 Lys TyrLys Ile Ala Gly Gly Ile Ala Gly Gly Leu Ala Leu Leu 1 5 10 15 425 15 PRTArtificial Sequence Pf EXP1 82 (peptide 1188.13) 425 Ala Gly Leu Leu GlyAsn Val Ser Thr Val Leu Leu Gly Gly Val 1 5 10 15 426 15 PRT ArtificialSequence Pf LSA1 94 (peptide 27.0408) 426 Gln Thr Asn Phe Lys Ser LeuLeu Arg Asn Leu Gly Val Ser Glu 1 5 10 15 427 15 PRT Artificial SequencePf SSP2 165 (peptide 35.0171) 427 Pro Asp Ser Ile Gln Asp Ser Leu LysGlu Ser Arg Lys Leu Asn 1 5 10 15 428 15 PRT Artificial Sequence Pf SSP2211 (peptide 35.0172) 428 Lys Cys Asn Leu Tyr Ala Asp Ser Ala Trp GluAsn Val Lys Asn 1 5 10 15 429 10 PRT Artificial Sequence Pf SSP2 14(peptide 1167.21) 429 Phe Leu Ile Phe Phe Asp Leu Phe Leu Val 1 5 10 4309 PRT Artificial Sequence Pf CSP 425 (peptide 1167.08) 430 Gly Leu IleMet Val Leu Ser Phe Leu 1 5 431 9 PRT Artificial Sequence Pf EXP1 80(peptide 1167.12) 431 Val Leu Ala Gly Leu Leu Gly Asn Val 1 5 432 9 PRTArtificial Sequence Pf EXP1 2 (peptide 1167.13) 432 Lys Ile Leu Ser ValPhe Phe Leu Ala 1 5 433 9 PRT Artificial Sequence Pf EXP1 83 (peptide1167.10) 433 Gly Leu Leu Gly Asn Val Ser Thr Val 1 5 434 10 PRTArtificial Sequence Pf CSP 7 (peptide 1167.18) 434 Ile Leu Ser Val SerSer Phe Leu Phe Val 1 5 10 435 10 PRT Artificial Sequence Pf EXP1 91(peptide 1167.19) 435 Val Leu Leu Gly Gly Val Gly Leu Val Leu 1 5 10 4369 PRT Artificial Sequence Pf SSP2 511 (peptide 1167.36) 436 Leu Ala CysAla Gly Leu Ala Tyr Lys 1 5 437 9 PRT Artificial Sequence Pf LSA1 94(peptide 1167.32) 437 Gln Thr Asn Phe Lys Ser Leu Leu Arg 1 5 438 10 PRTArtificial Sequence Pf CSP 375 (peptide 1167.43) 438 Val Thr Cys Gly AsnGly Ile Gln Val Arg 1 5 10 439 9 PRT Artificial Sequence Pf EXP1 10(peptide 1167.24) 439 Ala Leu Phe Phe Ile Ile Phe Asn Lys 1 5 440 9 PRTArtificial Sequence Pf LSA 1 105 (peptide 1167.28) 440 Gly Val Ser GluAsn Ile Phe Leu Lys 1 5 441 10 PRT Artificial Sequence Pf LSA1 59(peptide 1167.47) 441 His Val Leu Ser His Asn Ser Tyr Glu Lys 1 5 10 44210 PRT Artificial Sequence Pf SSP2 510 (peptide 1167.51) 442 Leu Leu AlaCys Ala Gly Leu Ala Tyr Lys 1 5 10 443 10 PRT Artificial Sequence PfLSA1 11 (peptide 1167.46) 443 Phe Ile Leu Val Asn Leu Leu Ile Phe His 15 10 444 9 PRT Artificial Sequence Pf SHEBA 77 (peptide 1101.03) 444 MetPro Leu Glu Thr Gln Leu Ala Ile 1 5 445 10 PRT Artificial Sequence PfSSP2 539 (peptide 1167.61) 445 Thr Pro Tyr Ala Gly Glu Pro Ala Pro Phe 15 10 446 9 PRT Artificial Sequence Pf SSP2 14 (peptide 1167.14) 446 PheLeu Ile Phe Phe Asp Leu Phe Leu 1 5 447 9 PRT Artificial Sequence PfSSP2 230 (peptide 1167.16) 447 Phe Met Lys Ala Val Cys Val Glu Val 1 5448 9 PRT Artificial Sequence Pf SSP2 15 (peptide 1167.15) 448 Leu IlePhe Phe Asp Leu Phe Leu Val 1 5 449 9 PRT Artificial Sequence Pf SSP2 51(peptide 1167.17) 449 Leu Leu Met Asp Cys Ser Gly Ser Ile 1 5 450 9 PRTArtificial Sequence Pf EXP1 91 (peptide 1167.09) 450 Val Leu Leu Gly GlyVal Gly Leu Val 1 5 451 8 PRT Artificial Sequence Pf SSP2 126 (peptide19.0051) 451 Leu Pro Tyr Gly Arg Thr Asn Leu 1 5 452 10 PRT ArtificialSequence Pf LSA1 1794 (peptide 16.0245) 452 Phe Gln Asp Glu Glu Asn IleGly Ile Tyr 1 5 10 453 9 PRT Artificial Sequence Pf CSP 15 (peptide16.0040) 453 Phe Val Glu Ala Leu Phe Gln Glu Tyr 1 5 454 9 PRTArtificial Sequence Pf LSA1 9 (peptide 1167.54) 454 Phe Tyr Phe Ile LeuVal Asn Leu Leu 1 5 455 9 PRT Artificial Sequence Pf EXP1 73 (peptide1167.53) 455 Lys Tyr Lys Leu Ala Thr Ser Val Leu 1 5 456 9 PRTArtificial Sequence Pf SSP2 8 (peptide 1167.56) 456 Lys Tyr Leu Val IleVal Phe Leu Ile 1 5 457 9 PRT Artificial Sequence Pf LSA1 1663 (peptide15.0184) 457 Leu Pro Ser Glu Asn Glu Arg Gly Tyr 1 5 458 9 PRTArtificial Sequence Pf SSP2 207 (peptide 16.0130) 458 Pro Ser Asp GlyLys Cys Asn Leu Tyr 1 5 459 9 PRT Artificial Sequence Pf LSA1 1664(peptide 16.0077) 459 Pro Ser Glu Asn Glu Arg Gly Tyr Tyr 1 5 460 9 PRTArtificial Sequence Pf SSP2 528 (peptide 1167.57) 460 Pro Tyr Ala GlyGlu Pro Ala Pro Phe 1 5 461 9 PRT Artificial Sequence Pf LSA1 1671(peptide 1167.55) 461 Tyr Tyr Ile Pro His Gln Ser Ser Leu 1 5 462 9 PRTArtificial sequence HBV pol 538-546 sub (peptide 1090.77) 462 Tyr MetAsp Asp Val Val Leu Gly Val 1 5 463 9 PRT Artificial sequence HIV 1 pol87(peptide 25.0062) Lys Leu Val Gly Lys Leu Asn Trp Ala 1 5

What is claimed is:
 1. An expression system which comprises a promoteroperably linked to a nucleotide sequence which encodes a peptidecomprising a first amino acid sequence which is a majorhistocompatibility (MHC) targeting sequence fused to a second amino acidsequence encoding one Class I MHC restricted CTL peptide epitope and oneuniversal HTL peptide epitope, wherein the CTL peptide epitope isselected from the group consisting of the HIV peptides set forth as SEQID NOs: 101, 105, 324-412, and
 463. 2. An expression system whichcomprises a promoter operably linked to a nucleotide sequence whichencodes a peptide comprising a first amino acid sequence which is an MHCtargeting sequence fused to a second amino acid sequence encoding oneClass I MHC restricted CTL peptide epitope and one universal HTL peptideepitope, wherein the CTL peptide epitope is selected from the groupconsisting of an analog of an HIV peptide set forth as SEQ ID NOs: 101,105, 324-412, and
 463. 3. A method to induce an immune response in asubject which comprises administering to a mammalian subject anexpression system, wherein the expression system comprises a promoteroperably linked to a nucleotide sequence which encodes a peptidecomprising a first amino acid sequence which is a majorhistocompatibility (MHC) targeting sequence fused to a second amino acidsequence encoding one Class I MHC restricted CTL peptide epitope and oneuniversal HTL peptide epitope, and wherein the CTL peptide epitope isselected from the group consisting of the HIV peptides set forth as SEQID NOs: 101, 105, 324-412, and
 463. 4. A method to induce an immuneresponse in a subject which comprises administering to a mammaliansubject an expression system, wherein the expression system comprises apromoter operably linked to a nucleotide sequence which encodes apeptide comprising a first amino acid sequence which is a majorhistocompatibility (MHC) targeting sequence fused to a second amino acidsequence encoding one Class I MHC restricted CTL peptide epitope and oneuniversal HTL peptide epitope, and wherein the CTL peptide epitope isselected from the group consisting of an analog of the HIV peptides setforth as SEQ ID NOs: 101, 105, 324-412, and 463.